3-Hydroxy-3-methylglutaryl-CoA reductase polynucleotides in isoprenoid production

ABSTRACT

The present invention relates to an isolated DNA sequence encoding an enzyme in the mevalonate pathway or the pathway from isopentenyl pyrophosphate to farnesyl pyrophosphate (i.e., HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, mevalonate pyrophosphate decarboxylase, and FPP synthase). Vectors and plasmids including such DNA, are also set forth. The invention also includes host cells transformed by such DNAs, or vectors or plasmids containing such DNAs. A process for the production of isoprenoids and carotenoids using such transformed host cells is also provided.

FIELD OF THE INVENTION

The present invention relates to the manufacture of isoprenoids usingmolecular biology techniques. In particular, the present inventionprovides DNAs, vectors and host cells for the efficient production ofvarious enzymes in the mevalonate pathway or for converting isopentylpyrophosphate to farnesyl pyrophosphate synthase.

BACKGROUND OF THE INVENTION

Astaxanthin is reportedly distributed in a wide variety of organismssuch as animals (e.g., birds, such as flamingo and scarlet ibis; fish,such as rainbow trout and salmon), algae and microorganisms. It is alsoreported that astaxanthin has a strong antioxidation property againstoxygen radicals, which is believed to be pharmaceutically useful forprotecting living cells against some diseases such as a cancer.Moreover, from a commercial prospective, there is an increasing demandfor astaxanthin as a coloring reagent especially in the fish farmingindustry, such as salmon farming, because astaxanthin imparts adistinctive orange-red coloration to the fish and contributes toconsumer appeal.

Phaffia rhodozyma is known as a carotenogenic yeast strain whichproduces astaxanthin specifically. Different from the othercarotenogenic yeast, Rhodotorula species, such as Phaffia rhodozyma (P.rhodozyma) can ferment some sugars such as D-glucose. This is acommercially important feature. In a recent taxonomic study, the sexualcycle of P. rhodozyma was revealed and its telemorphic state wasdesignated as Xanthophyllomyces dendrorhous (W. I. Golubev; Yeast 11,101-110, 1995). Some strain improvement studies to obtainhyper-producers of astaxanthin from P. rhodozyma have been conducted,but such efforts have been restricted to conventional methods includingmutagenesis and protoplast fusion in this decade.

Recently, Wery et al. reportedly developed a host vector system using P.rhodozyma in which a non-replicable plasmid was integrated into thegenome of P. rhodozyma at the locus of a ribosomal DNA in multiplecopies (Wery et al., Gene, 184, 89-97, 1997). Verdoes et al. reportedvectors for obtaining a transformant of P. rhodozyma, as well as itsthree carotenogenic genes which code for the enzymes that catalyze thereactions from geranylgeranyl pyrophosphate to β-carotene (Internationalpatent WO97/23633).

It has been reported that the carotenogenic pathway from a generalmetabolite, acetyl-CoA consists of multiple enzymatic steps incarotenogenic eukaryotes as shown in FIG. 1. In this pathway, twomolecules of acetyl-CoA are condensed to yield acetoacetyl-CoA which isconverted to 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) by the action of3-hydroxymethyl-3-glutaryl-CoA synthase. Next,3-hydroxy-3-methylglutaryl-CoA reductase converts HMG-CoA to mevalonate,to which two molecules of phosphate residues are then added by theaction of two kinases (mevalonate kinase and phosphomevalonate kinase).Mevalonate pyrophosphate is then decarboxylated by the action ofmevalonate pyrophosphate decarboxylase to yield isopentenylpyrophosphate (IPP) which becomes a building unit for a wide variety ofisoprene molecules which are necessary in living organisms. This pathwayis designated the “mevalonate pathway” taken from its importantintermediate, mevalonate.

In this pathway, IPP is isomerized to dimethylaryl pyrophosphate (DMAPP)by the action of IPP isomerase. Then, IPP and DMAPP are converted to aC₁₀ unit, geranyl pyrophosphate (GPP) by a head to tail condensation. Ina similar condensation reaction between GPP and IPP, GPP is converted toa C₁₅ unit, farnesyl pyrophosphate (FPP) which is an important substrateof cholesterol in animals, of ergosterol in yeast, and of thefarnesylation of regulation proteins, such as the RAS protein. Ingeneral, the biosynthesis of GPP and FPP from IPP and DMAPP arecatalyzed by one enzyme called FPP synthase (Laskovics et al.,Biochemistry, 20, 1893-1901, 1981).

On the other hand, in prokaryotes such as eubacteria, isopentenylpyrophosphate is reportedly synthesized in a different pathway via1-deoxyxylulose-5-phosphate from pyruvate which is absent in yeast andanimals (Rohmer et al., Biochem. J., 295, 517-524, 1993).

SUMMARY OF THE INVENTION

In studies of cholesterol biosynthesis, it was shown that therate-limiting steps of cholesterol metabolism were in the steps of thismevalonate pathway, especially in its early steps catalyzed by HMG-CoAsynthase and HMG-CoA reductase. It was recognized in accordance with thepresent invention that the biosynthetic pathways of cholesterol andcarotenoid which share their intermediate pathway from acetyl-CoA to FPPcan be used to improve the rate-limiting steps in the carotenogenicpathway. These steps may exist in the steps of mevalonate pathway,especially in the early mevalonate pathway such as the steps catalyzedby HMG-CoA synthase and HMG-CoA reductase. Improved yields ofcarotenoids, especially astaxanthin, are achievable using the process ofthe present invention.

In accordance with this invention, the genes and the enzymes involved inthe mevalonate pathway from acetyl-CoA to FPP which are biologicalmaterials useful in improving the astaxanthin production process areprovided. In the present invention, cloning and determination of thegenes which code for HMG-CoA synthase, HMG-CoA reductase, mevalonatekinase, mevalonate pyrophosphate decarboxylase and FPP synthase isprovided.

This invention also relates to the characterization of such enzymes as aresult of the expression of such genes in suitable host organisms suchas E. coli. These genes may be amplified in a suitable host, such as P.rhodozyma. The effects on carotenogenesis by these enzymes can beconfirmed by cultivation of such a transformant in an appropriate mediumunder appropriate cultivation conditions.

In one embodiment, there is provided an isolated DNA sequence coding forat least one enzyme involved in the mevalonate pathway or the reactionpathway from isopentenyl pyrophosphate to farnesyl pyrophosphate. Morespecifically, such enzymes in accordance with the present invention arethose having, for example, the following activities:3-hydroxy-3-methylglutaryl-CoA synthase activity,3-hydroxy-3-methylglutaryl-CoA reductase activity, mevalonate kinaseactivity, mevalonate pyrophosphate decarboxylase activity and farnesylpyrophosphate synthase.

The isolated DNA sequences according to the present invention are morespecifically characterized in that (a) they code for enzymes havingamino acid sequences as set forth in SEQ ID NOs: 6, 7, 8, 9 and 10. TheDNA sequences may alternatively (b) code for variants of such enzymesselected from (i) allelic variants and (ii) enzymes having one or moreamino acid addition, insertion, deletion and/or substitution and havingthe stated enzyme activity.

Preferably, the isolated DNA sequence defined above is derived from agene of Phaffia rhodozyma. Such a DNA sequence is represented in SEQ IDNOs: 1, 2, 4 and 5. This DNA sequence may also be an isocoding or anallelic variant for the DNA sequence represented in SEQ ID NOs: 1, 2, 4and 5. In addition, this DNA sequence can be a derivative of a DNAsequence represented in SEQ ID NOs: 1, 2, 4 and 5 with addition,insertion, deletion and/or substitution of one or more nucleotide(s),and coding for a polypeptide having the above-referenced enzymeactivity.

In the present invention, such derivatives can be made by recombinantmeans using one of the DNA sequences as disclosed herein by methodsknown in the art and disclosed, e.g. by Sambrook et al. (MolecularCloning, Cold Spring Harbor Laboratory Press, New York, USA, secondedition 1989) which is hereby incorporated by reference. Amino acidexchanges in proteins and peptides which do not generally alter theactivity of the protein or peptide are known in the art and aredescribed, for example, by H. Neurath and R. L. Hill in The Proteins(Academic Press, New York, 1979, see especially FIG. 6, page 14). Themost commonly occurring exchanges are: Ala/Ser, Val/Ile, Asp/Glu,Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro,Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly, as well as thesein reverse.

The present invention also provides an isolated DNA sequence coding fora polypeptide having mevalonate kinase activity, which DNA is selectedfrom (i) a DNA sequence represented in SEQ ID NO: 3; (ii) an isocodingor an allelic variant for the DNA sequence represented in SEQ ID NO: 3;and (iii) a derivative of a DNA sequence represented in SEQ ID NO: 3with addition, insertion, deletion and/or substitution of one or morenucleotide(s).

The present invention is intended to include those DNA sequences asspecified above and as disclosed in the sequence listing, as well astheir complementary strands, DNA sequences which include thesesequences, DNA sequences which hybridize under standard conditions withsuch sequences or fragments thereof and DNA sequences, which because ofthe degeneracy of the genetic code, do not hybridize under standardconditions with such sequences, but which code for polypeptides havingexactly the same amino acid sequence.

For purposes of the present invention, “standard conditions” forhybridization mean the conditions which are generally used by a oneskilled in the art to detect specific hybridization signals and whichare described, e.g. by Sambrook et al., supra. Preferably, the standardconditions are so called “stringent hybridization” and non-stringentwashing conditions, more preferably so called stringent hybridizationand stringent washing conditions. The stringency (high vs. low) of aparticular hybridization will of course vary depending upon, forexample, the salt concentration and temperature of the hybridization andwashes, as well as the lengths of the probe and target DNAs. Theexamples provided herein set forth representative hybridizationconditions but are not to be construed as limiting the scope of theinvention.

Furthermore, DNA sequences which can be made by the polymerase chainreaction using primers designed on the basis of the DNA sequencesdisclosed herein by methods known in the art are also included in thepresent invention. It is understood that the DNA sequences of thepresent invention may also be made synthetically as described, e.g. inEP 747 483 which is hereby incorporated by reference.

Another embodiment of the present invention is a recombinant DNA,preferably a vector and/or a plasmid including a sequence coding for anenzyme functional in the mevalonate pathway or the reaction pathway fromisopentenyl pyrophosphate to farnesyl pyrophosphate. The recombinant DNAvector and/or plasmid of the present invention includes regulatoryregions, such as for example, promoters and terminators, as well as openreading frames of above named DNAs.

Another embodiment of the present invention is a process fortransforming a host organism with a recombinant DNA, vector or plasmid.The recombinant organism of the present invention overexpresses a DNAsequence encoding an enzyme involved in the mevalonate pathway or thereaction pathway from isopentenyl pyrophosphate to farnesylpyrophosphate. The host organism transformed with the recombinant DNA isintended to be used for, e.g., producing isoprenoids and carotenoids, inparticular astaxanthin. Thus the present invention also includes suchrecombinant organisms/transformed hosts.

Another embodiment of the present invention is a method for theproduction of isoprenoids or carotenoids, preferably carotenoids, whichincludes cultivating recombinant organisms containing a DNA constructcoding for such isoprenoids or carotenoids.

Another embodiment of the present invention is a method for producing anenzyme involved in the mevalonate pathway or the reaction pathway fromisopentenyl pyrophosphate to farnesyl pyrophosphate. This methodincludes culturing a recombinant organism as mentioned above, underconditions conducive to the production of the enzyme. The method mayalso relate to obtaining the purified enzyme itself.

Another embodiment is a process for overexpressing an enzyme in themevalonate pathway or an enzyme in the pathway for convertingisopentenyl pyrophosphate to farnesyl pyrophosphate. This processincludes selecting at least one DNA sequence from the group consistingof SEQ ID NOs: 1-5; transforming a host cell culture with at least oneof the DNA sequences selected; expressing in the host cell at least oneenzyme in the mevalonate pathway or an enzyme in the pathway forconverting isopentenyl pyrophosphate to farnesyl pyrophosphate; andrecovering the enzyme(s) from the culture.

The present invention will be understood more easily on the basis of theenclosed figures and the more detailed explanations given below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a deduced biosynthetic pathway from acetyl-CoA toastaxanthin in P. rhodozyma.

FIG. 2 shows the expression study by using an artificial mvk geneobtained from an artificial nucleotide addition at the amino terminalend of a pseudo-mvk gene from P. rhodozyma. The cells from 50 ml ofbroth were subjected to 10% sodium dodecyl sulfide-polyacrylamide gelelectrophoresis (SDS-PAGE). Lane 1, E. coli (M15 (pREP4) (pQE30) withoutIPTG); Lane 2, E. coli (M15 (pREP4) (pQE30) with 1 mM IPTG); Lane 3,Molecular weight marker (105 kDa, 82.0 kDa, 49.0 kDa, 33.3 kD and 28.6kDa, up to down, BIO-RAD); Lane 4, E.coli (M15 (pREP4) (pMK1209 #3334)without IPTG); Lane 5, E.coli (M15 (pREP4) (pMK1209 #3334) with 1 mMIPTG).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an isolated DNA sequence which codes forenzymes which are involved in a biological pathway that includes themevalonate pathway or the reaction pathway from isopentenylpyrophosphate to farnesyl pyrophosphate. The enzymes of the presentinvention may be exemplified by those involved in the mevalonate pathwayor the reaction pathway from isopentenyl pyrophosphate to farnesylpyrophosphate in Phaffia rhodozyma. These sequences include, forexample, 3-hydroxy-3-methylglutaryl-CoA synthase,3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, mevalonatepyrophosphate decarboxylase and farnesyl pyrophosphate synthase.

The present invention has utility in the production of the compoundsinvolved in the mevalonate pathway and the carotenogenic pathway andvarious products derived from such compounds. The compounds involved inthe mevalonate pathway are acetoacetyl-CoA,3-hydroxymethyl-3-glutaryl-CoA, mevalonic acid, mevalonate-phosphate,mevalonate-pyrophosphate and isopentenyl-pyrophosphate. Subsequently,isopentenyl-pyrophosphate is converted to geranylgeranyl-pyrophosphatethrough geranyl-pyrophosphate and farnesyl-pyrophosphate via the“Isoprene Biosynthesis” reactions as indicated in FIG. 1.

The compounds involved in the carotenogenic pathway aregeranylgeranylpyrophosphate, phytoene, lycopene, β-carotene andastaxanthin. Among the compounds involved in the above-mentionedbiosynthesis, geranyl-pyrophosphate may be utilized for the productionof ubiquinone. Farnesyl-pyrophosphate may be utilized for the productionof sterols, such as cholesterol and ergosterol.Geranylgeranyl-pyrophosphate is used to produce vitamin K, vitamin E,chlorophyll and the like. Thus, the present invention has particularutility for the biological production of isoprenoids. As used herein,the term “isoprenoids” is intended to mean a series of compounds havingan isopentenyl-pyrophosphate as a skeleton unit. Further examples ofisoprenoids are vitamin A and vitamin D₃.

For purposes of the present invention, the term “DNA” is intended tomean a cDNA which contains only an open reading frame flanked betweenthe short fragments in its 5′- and 3′-untranslated region and a genomicDNA which also contains its regulatory sequences, such as its promoterand terminator which are necessary for the expression of the gene ofinterest.

In general, a gene consists of several parts which have differentfinctions from each other. In eukaryotes, genes which encode acorresponding protein are transcribed to premature messenger RNA(pre-mRNA) differing from the genes for ribosomal RNA (rRNA), smallnuclear RNA (snRNA) and transfer RNA (tRNA). Although RNA polymerase II(PolII) plays a central role in this transcription event, PolII cannotsolely start transcription without a cis element covering an upstreamregion containing a promoter and an upstream activation sequence (UAS)and a trans-acting protein factor.

At first, a transcription initiation complex which consists of severalbasic protein components recognizes the promoter sequence in the5′-adjacent region of the gene to be expressed. In this event, someadditional participants are required in the case of the gene which isexpressed under some specific regulation, such as a heat shock response,or adaptation to a nutrition starvation, etc. In such a case, a UAS isrequired to exist in the 5′-untranslated upstream region around thepromoter sequence, and some positive or negative regulator proteinsrecognize and bind to the UAS. The strength of the binding of thetranscription initiation complex to the promoter sequence is affected bysuch a binding of the trans-acting factor around the promoter. Thisenables regulation of the transcription activity.

After activation of a transcription initiation complex byphosphorylation, a transcription initiation complex initiatestranscription from the transcription start site. Some parts of thetranscription initiation complex are detached as an elongation complexwhich continues the transcription from the promoter region to the 3′direction of the gene (this step is called a promoter clearance event).The elongation complex continues transcription until it reaches atermination sequence that is located in the 3′-adjacent downstreamregion of the gene. Pre-mRNA thus generated is modified in the nucleusby the addition of a cap structure at the cap site which substantiallycorresponds to the transcription start site, and by the addition ofpolyA stretches at the polyA signal which is located at the 3′-adjacentdownstream region. Next, intron structures are removed from the codingregion and exon structures are combined to yield an open reading framewhose sequence corresponds to the primary amino acid sequence of acorresponding protein. This modification, in which a mature mRNA isgenerated, is necessary for stable gene expression.

As used herein, the term “cDNA” is intended to mean the DNA sequencewhich is reverse-transcribed from this mature mRNA sequence. It can besynthesized by the reverse transcriptase derived from viral species byusing a mature mRNA as a template, experimentally.

To express a gene which was derived from a eukaryote, a procedure inwhich a cDNA is cloned into an expression vector in, for example, E.coli, is often used as shown in this invention. This procedure is usedbecause intron structure specificity varies among eucaryotic organismswhich results in an inability to recognize the intron sequence from thatof other species. In fact, a prokaryote has no intron structure in itsown genetic background. Even in the yeast, the genetic background isdifferent between ascomycetea to which Saccharomyces cerevisiae belongsand basidiomycetea to which P. rhodozyma belongs. For example, Wery etal. showed that the intron structure of the actin gene from P. rhodozymacannot be recognized nor spliced by the ascomycetous yeast,Saccharomyces cerevisiae (Yeast, 12, 641-651, 1996).

It has been reported that the intron structures of some genes involveregulation of their gene expressions (Dabeva, M. D. et al., Proc. Natl.Acad. Sci. U.S.A., 83, 5854, 1986). Therefore, it may be important touse a genomic fragment which still contains its introns in the case ofself-cloning of a gene of interest whose intron structure involves sucha regulation of its own gene expression.

To apply a genetic engineering method for a strain improvement study, itis necessary to study its genetic mechanism during, e.g., transcriptionand translation. It is also important to determine the genetic sequenceof the gene, including for example, its UAS, promoter, intron structureand terminator in order to study the genetic mechanism.

According to this invention, the genes which code for the enzymes in themevalonate pathway were cloned from genomic DNA of P. rhodozyma. Thegenomic sequence containing the HMG-CoA synthase (hmc) gene, the HMG-CoAreductase (hmg) gene, the mevalonate kinase (mvk) gene, the mevalonatepyrophosphate decarboxylase (mpd) gene and the FPP synthase (fps) geneincluding their 5′- and 3′-adjacent regions, as well as their intronstructures were determined.

Initially, a partial gene fragment containing a portion of the hmc, hmg,mvk, mpd and fps genes was cloned using a degenerate PCR method. Usingthis degenerate PCR method, the gene of interest can be cloned which hasa high amino acid sequence homology to the known enzyme from otherspecies which has the same or similar function. A degenerate primer,which is used as a primer in degenerate PCR, was designed by reversetranslation of the amino acid sequence to the corresponding nucleotides(“degenerated”). In such a degenerate primer, a mixed primer whichconsists of any of A, C, G or T, or a primer containing inosine at anambiguous codon is generally used. In this invention, mixed primers wereused as degenerate primers to clone the genes set forth above. In thepresent invention, the PCR conditions used can be varied depending onthe primers used and genes cloned as described hereinafter.

An entire gene containing its coding region with its intron, as well asits regulation region, such as a promoter or a terminator can be clonedfrom a chromosome by screening a genomic library which is constructed ina vector such as a phage vector or a plasmid vector in an appropriatehost cell using as a labeled probe a partial DNA fragment obtained bydegenerate PCR as described above. In the present invention, a hoststrain such as E. coli, and a vector such as an E. coli vector, a phagevector such as a λ phage vector, or a plasmid vector such as a pUCvector are used in the construction of a library and the followinggenetic manipulations such as a sequencing, a restriction digestion, aligation and the like are carried out.

In this invention, an EcoRI genomic library of P. rhodozyma wasconstructed in the derivatives of λ vector, λZAPII and λDASHII dependingon the insert size. The insert size, i.e., the length of insert to becloned, was determined by Southern blot hybridization for each genebefore construction of a library. In this invention, the DNA probes werelabeled with digoxigenin (DIG), a steroid hapten instead of aconventional ³²P label, using the protocol which was prepared by thesupplier (Boehringer-Mannheim).

A genomic library constructed from the chromosome of P. rhodozyma wasscreened using a DIG-labeled DNA fragment which contained a portion ofthe gene of interest as a probe. Hybridized plaques were picked up andused for further study. In the case of λDASHII (insert size was from 9kb to 23 kb), prepared λDNA was digested by the restriction enzymeEcoRI, followed by cloning of the EcoRI insert into a plasmid vector,such as pUC19 or pBluescriptII SK+. When λZAPII was used in theconstruction of the genomic library, an in vivo excision protocol wasconveniently used for the succeeding step of cloning the insert fragmentinto the plasmid vector using a derivative of a single stranded M13phage, Ex assist phage (Stratagene). The plasmid DNA thus obtained wasthen sequenced.

In this invention, an automated fluorescent DNA sequencer (the ALFredsystem from Pharmacia) was used with an autocycle sequencing protocol inwhich the Taq DNA polymerase is employed in most cases of sequencing.

After determining the genomic sequence of each construct, the sequenceof the coding region was used for cloning a cDNA of the correspondinggene. The PCR method was also used to clone cDNA fragments. PCR primerswhose sequences were identical to the sequence at the 5′- and 3′-end ofthe open reading frame (ORF) were synthesized with the addition of anappropriate restriction site. Then, PCR was performed using those PCRprimers.

In this invention, a cDNA pool was used as a template for PCR cloning ofthe cDNA. The cDNA pool included various cDNA species which weresynthesized in vitro by viral reverse transcriptase and Taq polymerase(CapFinder Kit manufactured by Clontech was used) using the mRNAobtained from P. rhodozyma as a template. Using this procedure, acorresponding cDNA was obtained and its identity was confirmed by itssequence. Furthermore, the cDNA was used to confirm its enzyme activityafter cloning the cDNA fragment into an expression vector whichfunctions in E. coli, under the strong promoter activity of, forexample, the lac or T7 expression system.

Once enzyme activity of the expressed protein is confirmed, the proteinis purified and used to raise monoclonal and/or polyclonal antibodiesagainst the purified enzyme according to standard procedures in the art.These antibodies may be used to characterize the expression of thecorresponding enzyme in a strain improvement study, an optimizationstudy of the culture condition, and the like. Moreover, these antibodiesmay be used to purify large quantities of the enzyme in a single stepusing, for example, an affinity column.

In the present invention, after the rate-limiting step is determined inthe biosynthetic pathway which consists of multiple enzymatic reactions,three strategies can be used to enhance the enzymatic activity of therate-limiting reaction using its genomic sequence.

One strategy is to use the gene itself in its native form. The simplestapproach is to amplify the genomic sequence including its regulationsequence such as a promoter and a terminator. This is realized by thecloning of the genomic fragment encoding the enzyme of interest into theappropriate vector on which a selectable marker that finctions in P.rhodozyma is harbored.

A drug resistance gene which encodes the enzyme that enables the host tosurvive in the presence of a toxic antibiotic is often used for theselectable marker. The G418 resistance gene harbored in pGB-Ph9 (Wery etal. (Gene, 184, 89-97, 1997)) is an example of a drug resistance gene. Anutrition complementation marker may also be used in the host which hasan appropriate auxotrophy marker. The P. rhodozyma ATCC24221 strainwhich requires cytidine for its growth is one example of an auxotroph.By using CTP synthetase as donor DNA for ATCC24221, a host vector systemusing a nutrition complementation marker may be established.

In this system, two types of vectors may be used. One of the vectors isan integrated vector which does not have an autonomous replicatingsequence. pGB-Ph9 is an example of this type of a vector. Because such avector does not have an autonomous replicating sequence, it cannotreplicate by itself and is present only in an integrated form on thechromosome of the host as a result of a single-crossing recombinationusing the homologous sequence between the vector and the chromosome. Incase of increasing a dose of the integrated gene on the chromosome,amplification of the gene is often employed using a drug resistancemarker. By increasing the concentration of the corresponding drug in theselection medium, only the strain which contains the integrated genewill survive. Using such a selection method, a strain containing theamplified gene may be selected.

Another type of vector is a replicable vector which has an autonomousreplicating sequence. Such a vector can exist in a multicopy state whichin turn allows the harbored gene to also exist in a multicopy state. Byusing such a strategy, an enzyme of interest which is coded by theamplified gene can be overexpressed.

Another strategy to overexpress an enzyme of interest is to place thegene of interest under a strong promoter. In such a strategy, the genedoes not need to be in present a multicopy state. This strategy is alsoused to overexpress a gene of interest under the appropriate promoterwhose promoter activity is induced in an appropriate growth phase and atan appropriate time during cultivation. For example, production ofastaxanthin accelerates in the late phase of growth such as in the caseof production of a secondary metabolite. Thus, the expression ofcarotenogenic genes may be maximized during the late phase of growth. Insuch a phase, gene expression of most biosynthetic enzymes decreases.Thus, for example, by placing a gene involved in the biosynthesis of aprecursor of astaxanthin and whose expression is under the control of avegetative promoter, such as a gene which encodes an enzyme involved inthe mevalonate pathway, downstream of the promoter of the carotenogenicgenes, all the genes involved in the biosynthesis of astaxanthin becomesynchronized in their timing and phase of expression.

Another strategy to overexpress an enzyme of interest is to induce amutation in its regulatory elements. For this purpose, a kind ofreporter gene such as a β-galactosidase gene, a luciferase gene (a genecoding a green fluorescent protein), and the like is inserted betweenthe promoter and the terminator sequence of the gene of interest so thatall the parts including promoter, terminator and the reporter gene arefused and function with each other.

For example, transformed P. rhodozyma in which the reporter gene isintroduced on the chromosome or on the vector is mutagenized in vivo toinduce a mutation within the promoter region of the gene of interest.The mutation is monitored, for example, by detecting a change inactivity coded for by the reporter gene. If the mutation occurs in a ciselement of the gene, the mutation point would be determined by therescue of the mutagenized gene and subsequent sequencing. The determinedmutation is introduced to the promoter region on the chromosome byrecombination between a native promoter sequence and a mutated sequence.In the same procedure, the mutation occurring in the gene which encodesa trans-acting factor can be also obtained. It would also affect theoverexpression of the gene of interest.

A mutation may also be induced by in vitro mutagenesis of a cis elementin the promoter region. In this approach, a gene cassette, containing areporter gene fused to a promoter region derived from a gene of interestat its 5′-end and a terminator region from a gene of interest at its3′-end, is mutagenized and then introduced into P. rhodozyma. Bydetecting the difference in the activity of the reporter gene, aneffective mutation can be screened and identified. Such a mutation canbe introduced in the sequence of the native promoter region on thechromosome by the same methods used for in vivo mutation.

As a donor DNA, a gene which encodes an enzyme of the mevalonate pathwayor FPP synthase is introduced alone or co-introduced on a plasmidvector. A coding sequence which is identical to its native sequence, aswell as its allelic variant (a sequence which has one or more amino acidadditions, deletions and/or substitutions) can be used so long as itscorresponding enzyme has the stated enzyme activity. Such a vector isintroduced into P. rhodozyma by transformation and a transformant isselected by spreading the transformed cells on an appropriate selectionmedium such as, for example, YPD agar medium containing genetic in thecase of pGB-Ph9 as a vector or a minimal agar medium omitting cytidinewhen the auxotroph ATCC24221 is used as a recipient.

Such a genetically engineered P. rhodozyma is cultivated in anappropriate medium and evaluated for its production of astaxanthin. Ahyper-producer of astaxanthin thus selected may be confirmed in view ofthe relationship between its productivity and the level of gene orprotein expression which is introduced by such a genetic engineeringmethod.

Thus in the present invention, all three strategies may be used toenhance the enzymatic activity of the rate limiting step in theenzymatic pathways set forth above.

The following examples are set forth to illustrate compositions andprocesses of the present invention. These examples are provided forpurposes of illustration only and are not intended to be limiting in anysense.

EXAMPLES

The following materials and methods were employed in the examplesdescribed below:

Strains

P. rhodozyma ATCC96594 (This strain was redeposited on Apr. 8, 1998pursuant to the Budapest Treaty and was assigned accession No. 74438).

E. coli DH5α: F⁻, φ80d, lacZΔDM15, Δ(lacZYA-argF)U169, hsd(r_(K) ⁻,m_(K) ⁺), recA1, endA1, deoR, thi-1, supE44, gyrA96, relA1 (Toyobo)

E. coli XL1-Blue MRF′: Δ(mcrA)183, Δ(mcrCB-hsdSMR-mrr)173, endA1,supE44, thi-1, recA1, gyrA96, relA1, lac[F′ proAB, lacI^(q)ZΔM15, Tn10(tet^(r))] (Stratagene)

E. coli SOLR: e14⁻(mcrA), Δ(mcrCB-hsdSMR-mrr)171, sbcC, recB, recJ,umuC:: Tn5(kan^(r)), uvrC, lac, gyrA96, relA1, thi-1, endA1, λ^(R), [F′proAB, lacI^(q)Z ΔM15] Su⁻(nonsuppressing) (Stratagene, Calif., USA)

E. coli XL1 MRA (P2): Δ(mcrA)183, Δ(mcrCB-hsdSMR-mrr)173, endA1, supE44,thi-1, gyrA96, relA1, lac (P2 lysogen) (Stratagene)

E. coli BL21 (DE3) (pLysS): dcm⁻, ompTr_(B) ⁻m_(B) ⁻Ion⁻λ(DE3), pLysS(Stratagene)

E. coli M15 (pREP4)(QIAGEN)(Zamenhof P. J. et al., J. Bacteriol. 110,171-178, 1972)

E. coli KB822:pcnB80, zad:: Tn10, Δ(lacU169), hsdR17, endA1, thi-1,supE44

E. coli TOP10: F⁻, mcrA, Δ(mrr-hsdRMS-mcrBC), φ80, ΔlacZ M15, ΔlacX74,recA1, deoR, araD139, (ara-leu)7697, galU, galK, rpsL (Str^(r)), endA1,nupG (Invitrogen)

Vectors

λZAPII (Stratagene)

λDASHII (Stratagene)

pBluescriptIl SK+(Stratagene)

pUC57 (MBI Fermentas)

pMOSBlue T-vector (Amersham)

pET4c (Stratagene)

pQE30 (QIAGEN)

pCR2.1TOPO (Invitrogen)

Media

P. rhodozyma strain is maintained routinely in YPD medium (DIFCO). E.coli strain is maintained in LB medium (10 g Bacto-trypton, 5 g yeastextract (DIFCO) and 5 g NaCl per liter). NZY medium (5 g NaCl, 2 gMgSO₄—7H₂O, 5 g yeast extract (DIFCO), 10 g NZ amine type A (Sheffield)per liter) is used for phage propagation in a soft agar (0.7% agar(WAKO)). When an agar medium was prepared, 1.5% of agar (WAKO) wassupplemented.

Methods

General methods of molecular genetics were practiced according toMolecular Cloning: a Laboratory Manual, 2nd Edition (Cold Spring HarborLaboratory Press, 1989). Restriction enzymes and T4 DNA ligase werepurchased from Takara Shuzo (Japan).

Isolation of chromosomal DNA from P. rhodozyma was performed using aQIAGEN Genomic Kit (QIAGEN) following the protocol supplied by themanufacturer. Mini-preps of plasmid DNA from transformed E. coli wereperformed with the Automatic DNA isolation system (PI-50, Kurabo, Co.Ltd., Japan). Midi-preps of plasmid DNA from an E. coil transformantwere performed using a QIAGEN column (QIAGEN). Isolation of λDNA wasperformed with the Wizard lambda preps DNA purification system (Promega)following the protocol of the manufacturer. A DNA fragment was isolatedand purified from agarose using QIAquick or QIAEX II (QIAGEN).Manipulation of λ phage derivatives was done according to the protocolof the manufacturer (Stratagene).

Isolation of total RNA from P. rhodozyma was performed by the phenolmethod using Isogen (Nippon Gene, Japan). mRNA was purified from totalRNA thus obtained using a mRNA separation kit (Clontech). cDNA wassynthesized using a CapFinder CDNA construction kit (Clontech).

In vitro packaging was performed using Gigapack III gold packagingextract (Stratagene).

Polymerase chain reaction (PCR) was performed with a thermal cycler fromPerkin Elmer model 2400. Each PCR condition is described in theexamples. PCR primers were purchased from a commercial supplier orsynthesized with a DNA synthesizer (model 392, Applied Biosystems).Fluorescent DNA primers for DNA sequencing were purchased fromPharmacia. DNA sequencing was performed with the automated fluorescentDNA sequencer (ALFred, Pharmacia).

Competent cells of DH5 were purchased from Toyobo (Japan). Competentcells of M15 (pREP4) were prepared by the CaCl₂ method described bySambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory Press, 1989).

Example 1 Isolation of mRNA from P. rhodozyma and Construction of cDNALibrary

To construct a cDNA library of P. rhodozyma, total RNA was isolated byphenol extraction right after cell disruption. The mRNA from the P.rhodozyma ATCC96594 strain was purified using a mRNA separation kit(Clontech).

P. rhodozyma cells (ATCC96594 strain) from 10 ml of a two-day-culture inYPD medium were harvested by centrifugation (1500×g for 10 min.) andwashed once with extraction buffer (10 mM Na-citrate/HCl (pH 6.2)containing 0.7 M KCl). After suspending the cells in 2.5 ml ofextraction buffer, the cells were disrupted by a French presshomogenizer (Ohtake Works Corp., Japan) at 1500 kgf/cm² and immediatelymixed with 2× volumes of isogen (Nippon gene) according to the methodspecified by the manufacturer. In this step, 400 μg of total RNA wasrecovered.

This total RNA was purified using a mRNA separation kit (Clontech)according to the method specified by the manufacturer. Using thismethod, 16 μg of mRNA from P. rhodozyma ATCC96594 strain was obtained.

To construct a cDNA library, a CapFinder PCR cDNA construction kit(Clontech) was used according to the method specified by themanufacturer. One μg of purified mRNA was applied for a first strandsynthesis followed by PCR amplification. After this PCR amplification, 1mg of a cDNA pool was obtained.

Example 2 Cloning of the Partial hmc (3-hydroxy-3-methylglutaryl-CoAsynthase) Gene from P. rhodozyma

To clone a partial hmc gene from P. rhodozyma, a degenerate PCR methodwas exploited. Two mixed primers whose nucleotide sequences weredesigned and synthesized (as shown in TABLE 1) based on the commonsequence of known HMG-CoA synthase genes from other species.

Table 1 Sequence of Primers Used in the Cloning of the hmc Gene

Hmgs1; GGNAARTAYACNATHGGNYTNGGNCA (sense primer) (SEQ ID NO: 11)

Hmgs3; TANARNSWNSWNGTRTACATRTTNCC (antisense primer) (SEQ ID NO: 12)

(N=A, C, G or T; R=A or G, Y=C or T, H=A, T or C, S=C or G, W=A or T)

After a PCR reaction of 25 cycles at 95° C. for 30 seconds, 50° C. for30 seconds and 72° C. for 15 seconds using ExTaq (Takara Shuzo) as a DNApolymerase and the cDNA pool obtained in example 1 as a template, theresulting reaction mixture was separated by electrophoresis on anagarose gel. A PCR band that has the desired length was recovered andpurified by QIAquick (QIAGEN) according to the method of themanufacturer and then ligated to pMOSBlue T-vector (Amersham). Aftertransformation of competent E. coli DH5α cells with the isolated DNA, 6white colonies were selected and plasmids were isolated with anAutomatic DNA isolation system (Kurabo). As a result of sequencing, itwas found that the clone had a sequence whose deduced amino acidsequence was similar to known hmc genes. This isolated cDNA clone wasdesignated as pHMC211 and used for further study.

Example 3 Isolation of Genomic DNA from P. rhodozyma

To isolate a genomic DNA from P. rhodozyma, a QIAGEN genomic kit wasused according to the method specified by the manufacturer.

At first, the P. rhodozyma ATCC96594 strain cells from 100 ml of anovernight culture in YPD medium were harvested by centrifugation (1500×gfor 10 min.) and washed once with TE buffer (10 mM Tris/HCl (pH 8.0)containing 1 mM EDTA). After suspending the cells in 8 ml of Y1 bufferof the QIAGEN genomic kit, lyticase (SIGMA) was added at theconcentration of 2 mg/ml to disrupt the cells by enzymatic degradation.This reaction mixture was incubated for 90 minutes at 30° C. and thenproceeded to the next extraction step. Finally, 20 μg of genomic DNA wasobtained.

Example 4 Southern Blot Hybridization Using pHMC211 as a Probe

Southern blot hybridization was performed to clone a genomic fragmentwhich contains the hmc gene from P. rhodozyma. Two μg of genomic DNAfrom example 3 was digested by EcoRI and subjected to agarose gelelectrophoresis followed by acidic and alkaline treatment. The denaturedDNA was transferred to a nylon membrane (Hybond N+, Amersham) using atransblot (Joto Rika) apparatus for an hour. The DNA which wastransferred to the nylon membrane was fixed thereto with heat (80° C.,90 minutes). A probe was prepared by labeling template DNA (EcoRI- andSalI-digested pHMC211) using the DIG multipriming method (BoehringerMannheim).

Hybridization was performed with the method specified by themanufacturer (Boehringer Mannheim). The hybridization experiment wasperformed using a commercially available DIG (digoxigenin) labeling kitand luminescent detection kit (Boehringer Mannheim, Mannheim, Germany).Standard hybridization conditions were used as follows: Thehybridization solution contained formamide (WAKO) 50% (V/V), blockingreagent (Boehringer Mannheim) 2% (W/V), 5×SSC, N-lauroylsarcosine 0.1%(W/V), and SDS 0.3% (W/V). The hybridization was performed at 42° C.overnight. Washing and luminescent detection was performed according tothe protocol supplied by the manufacturer. For example, the followingstandard post hybridization washing routine may be used: wash the nylonmembrane twice for 5 minutes each in 2×SSC and 0.1% SDS at roomtemperature followed by 2 washes of 15 minutes each in 0.1 SSC and 0.1%SDS at 68° C. under constant agitation. These washing conditions may bevaried as is known in the art depending upon the DNA, probe and intendedresult. In the present example, a hybridized band was visualized in therange from 3.5 to 4.0 kilobases (kb).

Example 5 Cloning of a Genomic Fragment Containing hmc Gene

Four μg of the genomic DNA from Example 3 was digested with EcoRI andsubjected to agarose gel electrophoresis. Then, DNAs whose length iswithin the range from 3.0 to 5.0 kb were recovered using a QIAEX II gelextraction kit (QIAGEN) according to the method specified by themanufacturer. The purified DNA was ligated to 1 μg of EcoRI-digested andCIAP (calf intestine alkaline phosphatase) -treated λZAPII (Stratagene)at 16° C. overnight, and packaged by Gigapack III gold packaging extract(Stratagene). The packaged extract was used to infect an E. coli XL1BlueMRF′ strain and over-laid with NZY medium poured onto LB agar medium.About 6000 plaques were screened using an EcoRI- and SalI-digestedpHMC211 fragment as a probe. Two plaques were hybridized to the labeledprobe and subjected to the in vivo excision protocol according to themethod specified by the manufacturer (Stratagene). It was found thatisolated plasmids had the same fragments in the opposite direction toeach other based on restriction analysis and sequencing. As a result ofsequencing, the obtained EcoRi fragment contained the same nucleotidesequence as that of the pHMC211 clone. One of these plasmids wasdesignated pHMC526 and used for further study. A complete nucleotidesequence was obtained by sequencing deletion derivatives of pHMC526, andsequencing with a primer-walking procedure.

Using these methods, it was determined that the insert fragment ofpHMC526 consists of 3,431 nucleotides that contained 10 complete exonsand one incomplete exon and 10 introns with about 1 kb of 3′-terminaluntranslated region.

Example 6 Cloning of Upstream Region of hmc Gene

Cloning of the 5′-region adjacent to the hmc gene was performed using aGenome Walker Kit (Clontech) because pHMC 526 does not contain its 5′end of hmc gene. As a first step, the PCR primers whose sequences wereshown in Table 2 were synthesized.

Table 2 Sequence of Primers Used in the Cloning of the 5′-regionAdjacent to the hmc Gene

Hmc21; GAAGAACCCCATCAAAAGCCTCGA (primary primer) (SEQ ID NO: 13)

Hmc22; AAAAGCCTCGAGATCCTTGTGAGCG (nested primer) (SEQ ID NO: 14)

Protocols for the genomic library construction and the PCR conditionswere the same as those specified by the manufacturer using the genomicDNA preparation obtained in Example 3 as a PCR template. The PCRfragments that had an EcoRV site at the 5′ end (0.45 kb), and that had aPvuII site at the 5′ end (2.7 kb) were recovered and cloned intopMOSBlue T-vector using E. coli DH5α as a host strain. By sequencing 5independent clones from both constructs, it was confirmed that the 5′region adjacent to the hmc gene was cloned and a small part (0.1 kb) ofan EcoRI fragment within its 3′ end was found. The clone obtained by thePvuII construct in the above experiment was designated as pHMCPv708 andused for further study.

Next, Southern blot analysis was performed using the method set forth inExample 4. The 5′-region adjacent to the hmc gene contained in the 3 kbEcoRI fragment was determined. After construction of a 2.5 to 3.5 kbEcoRI library in λZAPII, 600 plaques were screened and 6 positive cloneswere selected. As a result of the sequencing of these 6 clones, it wasfound that 4 clones within 6 positive plaques had the same sequence asthat of the pHMCPv708. One of those clones was named pHMC723 and wasused for further analysis.

The PCR primers whose sequences are set forth in TABLE 3 below weresynthesized and used to clone a small (0.1 kb) EcoRI fragment locatedbetween the 3.5 kb and 3.0 kb EcoRI fragments on the chromosome of P.rhodozyma.

Table 3 Sequence of Primers Used in Cloning the Small EcoRI Portion ofthe hmc Gene

Hmc30; AGAAGCCAGAAGAGAAAA (sense primer) (SEQ ID NO: 15)

Hmc31; TCGTCGAGGAAAGTAGAT (antisense primer) (SEQ ID NO: 16)

The PCR conditions used were the same as shown in Example 2. Anamplified fragment (0.1 kb in length) was cloned into pMOSBlue T-vectorand used to transform E. coli DH5α. Plasmids were prepared from 5independent white colonies and subjected to sequencing.

Using the sequence information, it was determined that the nucleotidesequence (4.8 kb) contained the hmc gene (SEQ ID NO: 1). The codingregion was 2,432 base pairs in length and consisted of 11 exons and 10introns. Introns were scattered throughout the coding region without 5′or 3′ bias. It was found also that the open reading frame consists of467 amino acids (SEQ ID NO: 6) whose sequence is strikingly similar tothe known amino acid sequence of HMG-CoA synthase gene from otherspecies (49.6% identity to HMG-CoA synthase from Schizosaccharomycespombe).

Example 7 Expression of hmc Gene in E. coli and Confirmation of itsEnzymatic Activity

The PCR primers whose sequences are set forth in TABLE 4 below weresynthesized to clone a cDNA fragment of the hmc gene.

Table 4 Sequence of Primers Used in the Cloning of cDNA of hmc Gene

Hmc25; GGTACCATATGTATCCTTCTACTACCGAAC (sense primer) (SEQ ID NO: 17)

Hmc26; GCATGCGGATCCTCAAGCAGAAGGGACCTG (antisense primer) (SEQ ID NO: 18)

The PCR conditions were as follows; 25 cycles at 95° C. for 30 seconds,55° C. for 30 seconds and 72° C. for 3 minutes. As a template, 0.1 μg ofthe cDNA pool obtained in Example 2 was used, and Pfu polymerase wasused as a DNA polymerase. An amplified 1.5 kb fragment was recovered andcloned in pT7Blue-3 vector (Novagen) using a perfectly blunt cloning kit(Novagen) according to the protocol specified by the manufacturer.

Six independent clones from white colonies of E. coli DH5α transformantswere selected and plasmids were prepared from those transformants. As aresult of restriction analysis, 2 clones were selected for furthercharacterization by sequencing. One clone has an amino acid substitutionat position 280 (from glycine to alanine) and the other clone has asubstitution at position 53 (from alanine to threonine). Alignment ofamino acid sequences derived from known hmc genes showed that thealanine and glycine residues at position 280 were observed in all thesequences from other species. This fact suggested that an amino acidsubstitution at position 280 would not affect its enzymatic activity.This clone (mutant at position 280) was selected and designated pHMC731for a succeeding expression experiment.

Next, a 1.5 kb fragment obtained by NdeI- and BamHI-digestion of pHMC731was ligated to pET11c (Stratagene) digested by the same pairs ofrestriction enzymes, and introduced into E. coli DH5α. As a result ofrestriction analysis, a plasmid that had a correct structure (pHMC818)was recovered. Then, competent E. coli BL21 (DE3) (pLysS) cells(Stratagene) were transformed with the plasmid (pHMC818), and one clonethat had a correct structure was selected for further study.

For an expression study, strain BL21 (DE3) (pLysS) (pHMC818) and avector control strain BL21 (DE3) (pLysS) (pET11c) were cultivated in 100ml of LB medium at 37° C. until an OD of 0.8 at 600 nm was reached(about 3 hours) in the presence of 100 μg/ml of ampicillin. Then, thebroth was divided into two samples of the same volume, and then 1 mM ofisopropyl β-D-thiogalactopyranoside (IPTG) was added to one sample(induced). Cultivation of both samples was continued for another 4 hoursat 37° C. Twenty five μl of broth was removed from induced- anduninduced-cultures of the hmc clone and the vector control cultures andsubjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis. It was confirmed that a protein whose size wassimilar to the deduced molecular weight based on the nucleotide sequence(50.8 kDa) was expressed only in the case of the clone that was harboredin pHMC818 with the induction.

Cells from 50 ml of broth were harvested by centrifugation (1500×g, 10minutes), washed once and suspended in 2 ml of hmc buffer (200 mMTris-HCl (pH 8.2)). The cells were disrupted by a French presshomogenizer (Ohtake Works) at 1500 kgf/cm² to yield a crude lysate.After centrifugation of the crude lysate, a supernatant fraction wasrecovered and used as a crude extract for enzymatic analysis. Only inthe case of the lysate from the induced clone (pHMC818), was a whitepellet spun down and recovered. An enzyme assay for3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase was performed usingthe photometric assay according to the method by Stewart et al. (J.Biol. Chem. 241(5), 1212-1221, 1966). In the crude extract, the activityof 3-hydroxy-3-methylglutaryl-CoA synthase was not detected. As a resultof SDS-PAGE analysis of the crude extract, an expressed protein bandthat was observed in expressed broth had disappeared. Subsequently, thewhite pellet that was recovered from the crude lysate of the inducedpHMC818 clone was solubilized with 8 M guanidine-HCl, and then subjectedto SDS-PAGE analysis. The expressed protein was recovered in the whitepellet. This suggested that the expressed protein forms an inclusionbody.

Next, an expression experiment in more mild conditions was conducted.Cells were grown in LB medium at 28° C. and the induction was performedby addition of 0.1 mM of IPTG. Subsequently, incubation was continuedfor another 3.5 hours at 28° C. and then the cells were harvested.Preparation of the crude extract was the same as the previous protocol.Their results are summarized in TABLE 5. It was shown that HMG-CoAsynthase activity was only observed in the induced culture of therecombinant strain harboring the hmc gene. This indicates that thecloned hmc gene encodes HMG-CoA synthase.

TABLE 5 Enzymatic characterization of hmc cDNA clone μmol of HMG-CoA/plasmid IPTG minute/mg-protein PHMC818 − 0 + 0.146 PET11c − 0 + 0

Example 8 Cloning of hmg (3-hydroxymethyl-3-glutaryl-CoA reductase) Gene

In this example, the cloning protocol for the hmg gene was substantiallythe same as the protocol used to clone the hmc gene shown in Examples 2to 7. At first, the PCR primers whose sequences are shown in TABLE 6were synthesized based on the common sequences of HMG-CoA reductasegenes from other species.

Table 6 Sequence of Primers Used in the Cloning of hmg Gene

Red1; GCNTGYTGYGARAAYGTNATHGGNTAYATGCC (sense primer) (SEQ ID NO: 19)

Red2; ATCCARTTDATNGCNGCNGGYTTYTTRTCNGT (antisense primer) (SEQ ID NO:20)

(N=A, C, G or T; R=A or G; Y=C or T, H=A, T or C, D=A, G or T)

After a PCR reaction of 25 cycles at 95° C. for 30 seconds, 54° C. for30 seconds and 72° C. for 30 seconds using ExTaq (Takara Shuzo) as a DNApolymerase, the reaction mixture was separated by electrophoresis on anagarose gel. A PCR band that had the desired length was recovered andpurified by QIAquick (QIAGEN) according to the manufacturer's method andthen ligated into pUC57 vector (MBI Fermentas). After the transformationof competent E. coli DH5α cells with this vector, 7 white colonies wereselected and plasmids were isolated from those transformants.

As a result of sequencing, it was found that all the clones had asequence whose deduced amino acid sequence was similar to known HMG-CoAreductase genes. One of the isolated cDNA clones was designated as pRED1219 and was used for further study.

Next, a genomic fragment containing 5′- and 3′-regions adjacent to thehmg gene was cloned with the Genome Walker kit (Clontech). The 2.5 kbfragment of 5′ adjacent region (pREDPVu1226) and the 4.0 kb fragment ofthe 3′ adjacent region of the hmg gene (pREDEVd1226) were cloned. Basedon the sequence of the insert of pREDPVu1226, PCR primers whosesequences are shown in TABLE 7 were synthesized.

Table 7 Sequence of Primers Used in the Cloning of cDNA of hmg Gene

Red8; GGCCATTCCACACTTGATGCTCTGC (antisense primer) (SEQ ID NO: 21)

Red9; GGCCGATATCTTTATGGTCCT (sense primer) (SEQ ID NO: 22)

Subsequently, a cDNA fragment containing a long portion of the hmg cDNAsequence was cloned by PCR using Red 8 and Red 9 as PCR primers and thecDNA pool prepared in Example 2 as template. The cloned plasmid wasdesignated pRED107. The PCR conditions were as follows; 25 cycles for 30seconds at 94° C., 30 seconds at 55° C. and 1 minute at 72° C.

A Southern blot hybridization study was performed to clone a genomicsequence which contains the entire hmg gene from P. rhodozyma. A probewas prepared by labeling a template DNA (pRED107) according to the DIGmultipriming method. Hybridization was performed with the methodspecified by the manufacturer. As a result, the labeled probe hybridizedto two bands that were 12 kb and 4 kb in length. As a result ofsequencing of pREDPVu1226, an EcoRI site was not found in the cloned hmgregion. This suggested that another species of hmg gene (that has 4 kbof hybridized EcoRI fragment) existed on the genome of P. rhodozyma asfound in other organisms.

Next, a genomic library consisting of 9 to 23 kb of an EcoRI fragment inthe λDASHII vector was constructed. The packaged extract was used toinfect E. coli XL1 Blue, MRA(P2) strain (Stratagene) and over-laid withNZY medium poured onto LB agar medium. About 5000 plaques were screenedusing the 0.6 kb fragment of Stul-digested pRED107 as a probe. 4 plaqueswere hybridized to the labeled probe. Then, a phage lysate was preparedand DNA was purified with the Wizard lambda purification systemaccording to the method specified by the manufacturer (Promega). Thepurified DNA was digested with EcoRI to isolate a 10 kb EcoRI fragmentwhich was cloned into an EcoRI-digested and CIAP-treated pBluescriptIIKS-(Stratagene). Eleven white colonies were selected and subjected to acolony PCR using Red9 and −40 universal primer (Pharmacia).

Template DNA for a colony PCR was prepared by heating a cell suspensionin which a picked-up colony was suspended in 10 μl of sterilized waterfor 5 minutes at 99° C. prior to a PCR reaction (PCR conditions; 25cycles for 30 seconds at 94° C., 30 seconds at 55° C. and 3 minutes at72° C.). One colony gave 4 kb of a positive PCR band. This indicatedthat the clone contained the entire hmg gene. A plasmid from thispositive clone was prepared and designated pRED611. Subsequently,deletion derivatives of pRED611 were made for sequencing. By combiningthe sequence obtained from the deletion mutants with the sequenceobtained by a primer-walking procedure, the nucleotide sequence of 6370base pairs which contains the hmg gene from P. rhodozyma was determined(SEQ ID NO: 2).

The hmg gene from P. rhodozyma consists of 10 exons and 9 introns. Thededuced amino acid sequence of 1,091 amino acids in length (SEQ ID NO:7) showed an extensive homology to known HMG-CoA reductase (53.0%identity to HMG-CoA reductase from Ustilago maydis).

Example 9 Expression of Carboxyl-terminal Domain of hmg Gene in E. coli

Some species of prokaryotes have soluble HMG-CoA reductases or relatedproteins (Lam et al., J. Biol. Chem. 267, 5829-5834, 1992). However, ineukaryotes HMG-CoA reductase is tethered to the endoplasmic reticulumvia an amino-terminal membrane domain (Skalnik et al., J. Biol. Chem.263, 6836-6841, 1988). In fungi (i.e., Saccharomyces cerevisiae and thesmut fungus, Ustilago maydis) and in animals, the membrane domain islarge and complex, containing seven or eight transmembrane segments(Croxen et al. Microbiol. 140, 2363-2370, 1994). In contrast, themembrane domains of plant HMG-CoA reductase proteins have only one ortwo transmembrane segments (Nelson et al. Plant Mol. Biol. 25, 401-412,1994). Despite the difference in the structure and sequence of thetransmembrane domain, the amino acid sequences of the catalytic domainare conserved across eukaryotes, archaebacteria and eubacteria.

Croxen et al. showed that the C-terminal domain of HMG-CoA reductasederived from the maize fungal pathogen, Ustilago maydis was expressed inactive form in E. coli (Microbiology, 140, 2363-2370, 1994). Theinventors of the present invention tried to express a C-terminal domainof HMG-CoA reductase from P. rhodozyma in E. coli to confirm itsenzymatic activity.

At first, the PCR primers whose sequences were shown in TABLE 8 weresynthesized to clone a partial cDNA fragment of the hmg gene. The senseprimer sequence corresponds to the sequence which starts from the 597thamino acid (glutamate) residue. The length of the protein and cDNA whichwas expected to be obtained was 496 amino acids and 1.5 kb,respectively.

Table 8 Sequence of Primers Used in the Cloning of a Partial cDNA of hmgGene

Red54; GGTACCGAAGAAATTATGAAGAGTGG (sense primer) (SEQ ID NO: 23)

Red55; CTGCAGTCAGGCATCCACGTTCACAC (antisense primer) (SEQ ID NO: 24)

The PCR conditions were as follows; 25 cycles at 95° C. for 30 seconds,55° C. for 30 seconds and 72° C. for 3 minutes. As a template, 0.1 μg ofthe cDNA pool obtained in Example 2 and as a DNA polymerase, ExTaqpolymerase were used. An amplified 1.5 kb fragment was recovered andcloned in pMOSBlue T-vector (Novagen). Twelve independent clones fromwhite colonies of E. coli DH5∀ transformants were selected and plasmidswere prepared from those transformants. As a result of restrictionanalysis, all the clones were selected for further characterization bysequencing. One clone did not have a single amino acid substitutionthroughout the coding sequence and was designated pRED908.

Next, a 1.5 kb fragment obtained by KpnI- and PstI-digestion of pRED908was ligated to pQE30 (QIAGEN), digested by the same pairs of restrictionenzymes, and transformed to E. coli KB822. As a result of therestriction analysis, a plasmid that had a correct structure (pRED1002)was recovered. Then, competent E. coli M15 (pREP4) cells (QIAGEN) weretransformed and one clone that had a correct structure was selected forfurther study.

For an expression study, strain M15 (pREP4) (pRED1002) and vectorcontrol strain M15 (pREP4) (pQE30) were cultivated in 100 ml of LBmedium at 30° C. until the OD at 600 nm reached 0.8 (about 5 hours) inthe presence of 25 μg/ml of kanamycin and 100 μg/ml of ampicillin. Then,the broth was divided into two samples of the same volume, and 1 mM ofIPTG was added to one sample (induced). Cultivation of both samplescontinued for another 3.5 hours at 30° C. Twenty five μl of the brothwas removed from induced- and uninduced-cultures of the hmg clone andvector control cultures and subjected to SDS-PAGE analysis. It wasconfirmed that the protein whose size was similar to the deducedmolecular weight based on the nucleotide sequence (52.4 kDa) wasexpressed only in the case of the clone that harbored pRED1002 with theinduction.

Cells from 50 ml of broth were harvested by centrifugation (1500×g, 10minutes), washed once and suspended in 2 ml of hmg buffer (100 mMpotassium phosphate buffer (pH 7.0) containing 1 mM of EDTA and 10 mM ofdithiothreitol). Cells were disrupted by a French press (Ohtake Works)at 1500 kgf/cm² to yield a crude lysate. After centrifugation of thecrude lysate, a supernatant fraction was recovered and used as a crudeextract for enzymatic analysis. Only in the case of the induced lysateof the pRED1002 clone, a white pellet was spun down and recovered. Anenzyme assay for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase wasperformed by the photometric assay according to the method by Servouseet al. (Biochem. J. 240, 541-547, 1986). In the crude extract, theactivity of 3-hydroxy-3-methylglutaryl-CoA synthase was not detected. Asa result of SDS-PAGE analysis for the crude extract, the expressedprotein band that was present in the expressed broth was not observed.Next, the white pellet recovered from the crude lysate of inducedpRED1002 clone was solubilized with an equal volume of 20% SDS, and thensubjected to SDS-PAGE analysis. An expressed protein was recovered inthe white pellet, which indicators that the expressed protein would forman inclusion body.

Next, the expression experiment was performed in more mild conditions.Cells were grown in LB medium at 28° C. and the induction was performedby the addition of 0.1 mM of IPTG Then, incubation was continued foranother 3.5 hours at 28° C. and then the cells were harvested.Preparation of the crude extract was the same as the previous protocol.Results are summarized in TABLE 9. It was shown that 30 times higherinduction was observed, and this suggested that the cloned hmg genecodes HMG-CoA reductase.

TABLE 9 Enzymatic characterization of hmg cDNA clone μmol of NADPH/Plasmid IPTG minute/mg-protein PRED1002 − 0.002 + 0.059 pQE30 − 0 + 0

Example 10 Cloning of Mevalonate Kinase (mvk) Gene

The cloning protocol for the mvk gene used in this example wassubstantially the same as the protocol for the hmc gene shown inExamples 2 to 7. At first, PCR primers whose sequence are shown in TABLE10, were synthesized based on the common sequences of the mevalonatekinase genes from other species.

Table 10

Sequence of Primers Used in the Cloning of mvk Gene

Mk1; GCNCCNGGNAARGTNATHYTNTTYGGNGA (sense primer) (SEQ ID NO: 25)

Mk2; CCCCANGTNSWNACNGCRTTRTCNACNCC (antisense primer) (SEQ ID NO: 26)

(N=A, C, G or T; R=A or G, Y=C or T, H=A, T or C, S=C or G, W=A or T)

After a PCR reaction of 25 cycles at 95° C. for 30 seconds, 46° C. for30 seconds and 72° C. for 15 seconds using ExTaq as a DNA polymerase,the reaction mixture was separated by electrophoresis on an agarose gel.A 0.6 kb PCR band whose length was expected to contain a partial mvkgene was recovered and purified by QIAquick according to the methodindicated by the manufacturer and then ligated to pMOSBlue T-vector.After transformation of competent E. coli DH5∀ cells with thisconstruct, 4 white colonies were selected and plasmids were isolated. Asa result of sequencing, it was found that one of the clones had asequence whose deduced amino acid sequence was similar to knownmevalonate kinase genes. This cDNA clone was named as pMK128 and wasused for further study.

Next, a partial genomic clone which contained the mvk gene was cloned byPCR. The PCR primers whose sequences are shown in TABLE 11, weresynthesized based on the internal sequence of pMK128.

Table 11 Sequence of Primers Used in the Cloning of Genomic DNAContaining mvk Gene

Mk5; ACATGCTGTAGTCCATG (sense primer) (SEQ ID NO: 27)

Mk6; ACTCGGATTCCATGGA (antisense primer) (SEQ ID NO: 28)

The PCR conditions were 25 cycles for 30 seconds at 94° C., 30 secondsat 55° C. and 1 minute at 72° C. The amplified 1.4 kb fragment wascloned into pMOSBlue T-vector. As a result of sequencing, it wasconfirmed that a genomic fragment containing the mvk gene which hadtypical intron structures could be obtained and this genomic clone wasdesignated pMK224.

A Southern blot hybridization study was performed to clone a genomicfragment which contained an entire mvk gene from P. rhodozyma. A probewas prepared by labeling a template DNA, pMK224 digested by NcoI withthe DIG multipriming method. Hybridization was performed with the methodspecified by the manufacturer. As a result, the labeled probe hybridizedto a 6.5 kb band.

Next, a genomic library consisting of a 5 to 7 kb EcoRI fragment wasconstructed in the 8ZAPII vector. The packaged extract was used toinfect E coli XL1Blue, MRF′ strain (Stratagene) and over-laid with NZYmedium poured onto LB agar medium. About 5000 plaques were screenedusing a 0.8 kb NcoI-fragment digested from pMK224 as a probe. Sevenplaques were hybridized to the labeled probe. Then a phage lysate wasprepared according to the method specified by the manufacturer(Stratagene) and in vivo excision was performed using E. coli XL1BlueMRF′ and SOLR strains. Fourteen white colonies were selected andplasmids were isolated from those selected transformants. Then, isolatedplasmids were digested by NcoI and subjected to Southern blothybridization with the same probe as the plaque hybridization. Theinsert fragments of all the plasmids hybridized to the probe. Thisindicated that a genomic fragment containing the mvk gene could becloned. A plasmid from one of the positive clones was prepared anddesignated as pMK701. About 3 kb of sequence was determined by theprimer walking procedure and it was revealed that the 5′ end of the mvkgene was not contained in pMK701.

Next, a PCR primer was synthesized which had the following sequence;

TTGTTGTCGTAGCAGTGGGTGAGAG (SEQ ID NO: 29).

This primer was used to clone the 5′-adjacent genomic region of the mvkgene with the Genome Walker Kit according to the method specified by themanufacturer (Clontech). A specific 1.4 kb PCR band was amplified andcloned into pMOSBlue T-vector. All of the transformants of DH5∀ selectedhad the expected length of the insert. Subsequent sequencing revealedthat the 5′-adjacent region of the mvk gene could be cloned. One of theclones was designated as pMKEVR715 and was used for further study. As aresult of Southern blot hybridization using the genomic DNA prepared inexample 3, the labeled pMKEVR715 construct hybridized to a 2.7 kb EcoRIband. Then, a genomic library in which EcoRI fragments from 1.4 to 3.0kb in length were cloned into 8ZAPII was constructed. This genomiclibrary was screened with a 1.0 kb EcoRI fragment from pMKEVR715.Fourteen positive plaques were selected from 5000 plaques and plasmidswere prepared from those plaques with the in vivo excision procedure.

The PCR primers whose sequences are shown in TABLE 12, taken from theinternal sequence of pMKEVR715 were synthesized to select a positiveclone with a colony PCR.

Table 12 PCR Primers Used for Colony PCR to Clone 5′-adjacent Region ofmvk Gene

Mk17; GGAAGAGGAAGAGAAAAG (sense primer) (SEQ ID NO: 30)

Mk18; TTGCCGAACTCAATGTAG (antisense primer) (SEQ ID NO: 31)

PCR conditions were as follows: 25 cycles for 30 seconds at 94° C., 30seconds at 50° C. and 15 seconds at 72° C. From all the candidatesexcept one clone, the positive 0.5 kb band was yielded. One of theclones was selected and designated pMK723 to determine the sequence ofthe upstream region of mvk gene. After sequencing the 3′-region ofpMK723 and combining it with the sequence of pMK701, the genomicsequence of the 4.8 kb fragment containing the mvk gene was determined.

The mvk gene consists of 4 introns and 5 exons (SEQ ID NO: 3). Thededuced amino acid sequence except 4 amino acids at the amino terminalend (SEQ ID NO: 8) showed an extensive homology to known mevalonatekinase (44.3% identity to mevalonate kinase from Rattus norvegicus).

Example 11 Expression of mvk Gene by the Introduction of 1 Base at theAmino Terminal Region

Although the amino acid sequence showed a significant homology to knownmevalonate kinase, an appropriate start codon for mvk gene could not befound. This result indicated that the cloned gene might be a pseudogenefor mevalonate kinase. To confirm this assumption, PCR primers whosesequences are shown in TABLE 13 were synthesized to introduce anartificial nucleotide which resulted in the generation of an appropriatestart codon at the amino terminal end.

Table 13 PCR Primers Used for the Introduction of a Nucleotide into mvkGene

Mk33; GGATCCATGAGAGCCCAAAAAGAAGA (sense primer) (SEQ ID NO: 32)

Mk34; GTCGACTCAAGCAAAAGACCAACGAC (antisense primer) (SEQ ID NO: 33)

The artificial amino terminal sequence thus introduced was as follows;NH2-Met-Arg-Ala-Gln. After the PCR reaction of 25 cycles at 95° C. for30 seconds, 55° C. for 30 seconds and 72° C. for 30 seconds using ExTaqpolymerase as a DNA polymerase, the reaction mixture was subjected toagarose gel electrophoresis. An expected 1.4 kb PCR band was amplifiedand cloned into the pCR2.1 TOPO vector. After transformation ofcompetent E. coli TOP10 cells, 6 white colonies were selected andplasmids were isolated. As a result of sequencing, it was found that oneclone had only one amino acid residue change (Asp to Gly change at 81stamino acid residue in SEQ ID NO: 8). This plasmid was named pMK1130#3334 and used for further study.

Then, the insert fragment of pMK1130 #3334 was cloned into pQE30. Thisplasmid was named pMK1209 #3334. After transformation of the expressionhost, M15 (pREP4), an expression study was conducted. The M15 (pREP4)(pMK1209 #3334) strain and vector control strain (M15 (pREP4) (pQE30))were inoculated into 3 ml of LB medium containing 100 μg/ml ofampicillin. After cultivation at 37° C. for 3.75 hours, the culturebroth was divided into two samples. 1 mM IPTG was added to one sample(induced) and incubation of all samples was continued for 3 hours. Cellswere harvested from 50 μl of broth by centrifugation and were subjectedto SDS-PAGE analysis. A protein which had an expected molecular weightof 48.5 kDa was induced by the addition of IPTG in the culture of M15(pREP4) (pMK1209 #3334) although no induced protein band was observed inthe vector control culture (FIG. 2). This result suggested that theactivated form of the mevalonate kinase protein could be expressed byartificial addition of one nucleotide at the amino terminal end.

Example 12 Cloning of the Mevalonate Pyrophosphate Decarboxylase (mpd)Gene

In this example, the cloning protocol for the mpd gene was substantiallythe same as used to clone the hmc gene shown in Examples 2 to 7. Atfirst, the PCR primers whose sequences are shown in TABLE 14 weresynthesized based on the common sequences of the mevalonatepyrophosphate decarboxylase gene from other species.

Table 14 Sequence of Primers Used in the Cloning of the mpd Gene

Mpd1; HTNAARTAYTTGGGNAARMGNGA (sense primer) (SEQ ID NO: 34)

Mpd2; GCRTTNGGNCCNGCRTCRAANGTRTANGC (antisense primer) (SEQ ID NO: 35)

(N=A, C, G or T; R=A or G, Y=C or T, H=A, T or C, M=A or C)

After the PCR reaction of 25 cycles at 95° C. for 30 seconds, 50° C. for30 seconds and 72° C. for 15 seconds using ExTaq as a DNA polymerase,the reaction mixture was subjected to agarose gel electrophoresis. A 0.9kb PCR band whose length was expected to contain a partial mpd gene wasrecovered and purified by QIAquick according to the method prepared bythe manufacturer and then ligated to pMOSBlue T-vector. Aftertransformation of competent E. coli DH5∀ cells, 6 white colonies wereselected and plasmids were isolated therefrom. Two of the 6 clones hadthe expected insert length. As a result of sequencing, it was found thatone of the clones had a sequence whose deduced amino acid sequence wassimilar to known mevalonate pyrophosphate decarboxylase genes. This cDNAclone was designated pMPD129 and was used for further study.

Next, a partial genomic fragment which contained the mpd gene was clonedby PCR. As a result of PCR (whose condition was the same as that of thecloning of a partial cDNA fragment), the amplified 1.05 kb fragment wasobtained and was cloned into pMOSBlue T-vector. As a result ofsequencing, it was confirmed that a genomic fragment containing the mpdgene which had typical intron structures had been obtained. This genomicclone was designated pMPD220.

A Southern blot hybridization study was performed to clone a genomicfragment which contained the entire mpd gene from P. rhodozyma. Theprobe was prepared by labeling a template DNA, pMPD220 digested by KpnI,using the DIG multipriming method. Hybridization was performed using themethod specified by the manufacturer. As a result, the probe hybridizedto a band that was 7.5 kb in length. Next, a genomic library containinga 6.5 to 9.0 kb EcoRI fragment in the 8ZAPII vector was constructed. Thepackaged extract was used to infect an E. coli XL1Blue, MRF′ strain andwas over-laid with NZY medium poured onto LB agar medium. About 6000plaques were screened using the 0.6 kb fragment of KpnI-digested pMPD220as a probe. 4 plaques were hybridized to the labeled probe. Then, aphage lysate was prepared according to the method specified by themanufacturer (Stratagene) and an in vivo excision was performed using E.coli XL1Blue MRF′ and SOLR strains. 3 white colonies derived from 4positive plaques were selected and plasmids were isolated from thoseselected transformants. Then, the isolated plasmids were subjected to acolony PCR method whose protocol was the same as that in example 8. PCRprimers whose sequences are shown in TABLE 14, depending on the sequencefound in pMPD129 were synthesized and used for a colony PCR.

Table 15 Sequence of Primers Used in the Colony PCR to Clone a Genomicmpd Clone

Mpd7; CCGAACTCTCGCTCATCGCC (sense primer) (SEQ ID NO: 36)

Mpd8; CAGATCAGCGCGTGGAGTGA (antisense primer) (SEQ ID NO: 37)

The PCR conditions were substantially the same as used in the cloning ofthe mvk gene; 25 cycles for 30 seconds at 94° C., 30 seconds at 50° C.and 10 seconds at 72° C. All the clones, except one, produced a positive0.2 kb PCR band. A plasmid was prepared from one of the positive clonesand the plasmid was designated pMPD701 and about 3 kb of its sequencewas determined by the primer walking procedure (SEQ ID NO: 4). The ORFconsisted of 401 amino acids (SEQ ID NO: 9) whose sequence was similarto the sequences of known mevalonate pyrophosphate decarboxylase (52.3%identity to mevalonate pyrophosphate decarboxylase fromSchizosaccaromyces pombe). Also determined was a 0.4 kb fragment fromthe 5′-adjacent region which was expected to include its promotersequence.

Example 13 Cloning of Farnesyl Pyrophosphate Synthase (fps) Gene

In this example, the cloning protocol for the fps gene was substantiallythe same as the protocol for cloning the hmc gene shown in Examples 2 to7. At first, the PCR primers whose sequences are shown in TABLE 16 weresynthesized based on the common sequences of the farnesyl pyrophosphatesynthase gene from other species.

Table 16 Sequence of Primers Used in the Cloning of fps Gene

Fps1; CARGCNTAYTTYYTNGTNGCNGAYGA (sense primer) (SEQ ID NO: 38)

Fps2; CAYTTRTTRTCYTGDATRTCNGTNCCDATYTT (antisense primer) (SEQ ID NO:39)

(N=A, C, G or T; R=A or G, Y=C or T, D=A, G or T)

After the PCR reaction of 25 cycles at 95° C. for 30 seconds, 54° C. for30 seconds and 72° C. for 30 seconds using ExTaq as a DNA polymerase,the reaction mixture was subjected to agarose gel electrophoresis. A PCRband that had the desired length (0.5 kb) was recovered and purified byQIAquick according to the method prepared by the manufacturer and thenligated to pUC57 vector. After transformation of competent E. coli DH5∀cells, 6 white colonies were selected and plasmids were then isolated.One of the plasmids which had the desired length of an insert fragmentwas sequenced. As a result, it was found that this clone had a sequencewhose deduced amino acid sequence was similar to known farnesylpyrophosphate synthase genes. This cDNA clone was named as pFPS107 andwas used for further study.

Next, a genomic fragment was cloned by PCR using the same primer set ofFps1 and Fps2. The same PCR conditions for the cloning of a partial cDNAwere used. A 1.0 kb band was obtained which was subsequently cloned andsequenced. This clone contained the same sequence as the pFPS107 andsome typical intron fragments. This plasmid was designated pFPS113 andwas used for a further experiment.

Then, a 5′- and 3′-adjacent region containing the fps gene was clonedaccording to the method described in Example 8. At first, the PCRprimers whose sequences are shown in TABLE 17 were synthesized.

Table 17 Sequences of Primers Used for a Cloning of Adjacent Region offps Gene

Fps7; ATCCTCATCCCGATGGGTGAATACT (sense for downstream cloning) (SEQ IDNO: 40)

Fps9; AGGAGCGGTCAACAGATCGATGAGC (antisense for upstream cloning) (SEQ IDNO: 41)

Amplified PCR bands were isolated and cloned into pMOSBlue T-vector. Asa result of sequencing, it was found that the 5′-adjacent region (2.5 kbin length) and the 3′-adjacent region (2.0 kb in length) were cloned.These plasmids were designated pFPSSTu117 and pFPSSTd117, respectively.After sequencing both plasmids, an ORF was found that consisted of 1068base pairs with 8 introns. The deduced amino acid sequence showed anextensive homology to known farnesyl pyrophosphate synthase from otherspecies. Based on the sequence determined, two PCR primers weresynthesized with the sequences shown in TABLE 17 to clone a genomic fpsclone and a cDNA clone for fps gene expression in E. coli.

Table 18 Sequences of Primers Used for a cDNA and Genomic fps Cloning

Fps27; GAATTCATATGTCCACTACGCCTGA (sense primer) (SEQ ID NO: 42)

Fps28; GTCGACGGTACCTATCACTCCCGCC (antisense primer) (SEQ ID NO: 43)

The PCR conditions were as follows; 25 cycles for 30 seconds at 94° C.,30 seconds at 50° C. and 30 seconds at 72° C. One cDNA clone that hadthe correct sequence was selected as a result of sequencing analysis ofthe clones obtained by PCR and was designated pFPS113. Next, a Southernblot hybridization study was performed to clone a genomic fragment whichcontained the entire fps gene from P. rhodozyma. The probe was preparedby labeling a template DNA, pFPS113 using the DIG multipriming method.As a result, the labeled probe hybridized to a band that was about 10kb.

Next, a genomic library consisting of 9 to 15 kb of an EcoRI fragmentwas constructed in a 8DASHII vector. The packaged extract was used toinfect E. coli XL1 Blue, MRA(P2) strain (Stratagene) and over-laid withNZY medium poured onto LB agar medium. About 10000 plaques were screenedusing the 0.6 kb fragment of SacI- digested pFPS113 as a probe. Eightplaques were hybridized to the labeled probe. Then, a phage lysate wasprepared according to the method specified by the manufacturer(Promega). All the plaques were subjected to a plaque PCR using Fps27and Fps28 primers.

Template DNA for a plaque PCR was prepared by heating 2 μl of a solutionof phage particles for 5 minutes at 99° C. prior to the PCR reaction.The PCR conditions were the same as that of the pFPS113 cloninghereinbefore. All the plaques gave a 2 kb positive PCR band. Thissuggested that these clones had an entire region containing the fpsgene. One of the 8DNAs that harbored the fps gene was digested withEcoRI to isolate a 10 kb EcoRI fragment which was cloned into anEcoRI-digested and CIAP-treated pBluescriptII KS- (Stratagene).

Twelve white colonies from transformed E. coli DH5∀ cells were selectedand plasmids were prepared from these clones and subjected to colony PCRusing the same primer sets of Fps27 and Fps28 and the same PCRconditions. A 2 kb positive band was yielded from 3 of 12 candidates.One clone was cloned and designated pFPS603. It was confirmed that thesequence of the fps gene which was previously determined from thesequence of pFPSSTu117 and pFPSStd117 was substantially correct althoughthere were some PCR errors. Finally, the nucleotide sequence wasdetermined of the 4,092 base pairs which contains the fps gene from P.rhodozyma (FIG. 3). An ORF which consisted of 355 amino acids with 8introns was found (SEQ ID NO: 5). The deduced amino acid sequence (SEQID NO: 10) showed an extensive homology to known FPP synthase (65%identity to FPP synthase from Kluyveromyces lactis).

The invention being thus described, it will be seen that the same may bevaried in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention and all suchmodifications are intended to be included within the scope of thefollowing claims.

43 1 4775 DNA Phaffia rhodozyma 5′UTR (1239)..(1240) EXPERIMENTAL 1catcgaagag agcgaagtga ttagggaagc cgaagaggca ctaacaacgt ggttgtatat 60gtgtgtttat gagtgttata tcgtcaagaa cgaagtccat tcatttagct agacagggag 120agagggagaa acgtacgggt ttaccctatt ggaccagtct aaagagagaa cgagagtttt 180tgggtcggtc acctgaagag tttgaacctc cacaagttta ttctagatta tttccggggg 240tatgtgaagg ataatgtcaa actttgtcca gattgaagaa ggcaagaaag gaaaggggcg 300aacgagagta tcgtcccatc tatgggtgac cagtcgacct tctgcatcgg cgatcccgag 360aatggaaggt tccgatggat cagaagtagg tttcctaagc tcaaacatag gtcattgcga 420gtgagataca tatgcagact gatatgctag tcaaaccgaa cgagatttct ctgtttgctt 480tcaaaaagac gaaccaacca tttcatgtcc aagatggcag gtccttcgat tctttgaagc 540tcctccctga tgcggacaga aaagaataaa aagtagacag actgtcaagt cgacagcgca 600agtttatcaa gctgagcgag aaaactcgaa cttacatacc ttggccgtca gttctgtaga 660ccaagcatcg gcctttcctc tttgcggcag gtgtacgcgt tggctcacca tcgtcactct 720cgtctcctga cccgttgctt tccttgacag cagtctgttc cacaggtttc tctaactgat 780aggtcccaac agcaaagata tctggatgtc tatgtgagaa ctctactgag tcggcagagt 840acaccgtatc gatataggcg agtgaggaag ctttgaaagg tgaagaagta gcgaaagatc 900atcagcgaat gaggactatg acaaaaaaga aattttcgta taatccactg gacaaatcac 960cttccatcgt gtcctccaag agggtttcgt ctgaaacgta aggacgaggt attgatagat 1020gattgacctt gagtacgcgg atggacaagg aacgagccca ctcccagggc tatgtaacac 1080cacacgtgac tccacttgaa ttgcggcaga taaacgaagt cttacgatcg gacgactttg 1140taaccattta gttatttacc cgtcttgttt tcttactttg atcgtcccat tttagacaca 1200aaaaaagaag ccagaagaga aaagaataaa acgtctaccg tgttctctcc gaattcttac 1260cacacccaca aaaccataca caatctcaat ctagatatcc agttatgtac acttctacta 1320ccgaacagcg acccaaagat gttggaattc tcggtatgga ggtatgttgt tcaattctgt 1380ttgtgttcaa tctttaatca tctttagtcg actgaccggt tcttcctttt tttttcttca 1440tcaaacaaaa caacccttct cgattcatgt catctttctt tccaatgcgc tactccttct 1500gtagatctac tttcctcgac gagtgcgtaa ctattctctc ttctgcattc tctctctatt 1560cccatgttcg atccctcgcc ctcatatggg cgactgtttc atctcttttg cttccgtcca 1620ttcttctttg atcttgttca ttttctacta atatctcccg acgcgaaata caacactgac 1680cgcgatttct ctcgatcagg ccatcgctca caaggatctc gaggcttttg atggggttcc 1740ttccggaaag tacaccatcg gtctcggcaa caacttcatg gccttcaccg acgacactga 1800ggacatcaac tcgttcgcct tgaacggtca gtctcttccg tttcagcaat cgacaggaaa 1860aaggcccaag cgcatctcac tgacaccttt ctccgttttg caattccatt tgattgttag 1920ctgtttccgg tcttctatca aagtacaacg ttgatcccaa gtcaatcggt cgaattgatg 1980tcggaactga gtccatcatt gacaagtcca aatctgtcaa gacagtcctt atggacttgt 2040tcgagtccca cggcaacaca gatattgagg gtatcgactc caagaatgcc tgctacggtt 2100ctaccgcggc cctgttcaat gccgtcaact ggatcgagtc atcctcttgg gacggaagaa 2160atgccattgt cttctgcgga gacattgcca tctacgccga gggtgctgcc cgacctgccg 2220gaggtgctgg tgcttgcgcc atcctcatcg gacccgacgc tcccgtcgtc ttcgagcgtg 2280agttccaatc cgtcattttc ttccacggca gcggctgaaa caacccttat ccgtcattct 2340catcaatcta gccgtccacg gaaacttcat gaccaacgct tgggacttct acaagcctaa 2400tctttcttcg tatgttcaaa ttttgaagtt tgcgcttggg agagtcttac actaattcgg 2460ggtgctcgta tccttcgaat cgtttgttgc tttatagtga atacgttcgt ctgcgcacct 2520cctatattta gtttttgatc aaatattgtc cattgaatta actctgaaac cttctcctcc 2580aaatagccca ttgtcgatgg acctctctcc gtcacttcct acgtcaacgc cattgacaag 2640gcctatgaag cttaccgaac aaagtatgcc aagcgatttg gaggacccaa gactaacggt 2700gtcaccaacg gacacaccga ggttgccggt gtcagtgctg cgtcgttcga ttaccttttg 2760ttccacaggt aagcgtcatc ttctgtattc tccttaaatt caaccgatca acggagttaa 2820ttcgtgtcat catattatct tgttggaaca gtccttacgg aaagcaggtt gtcaaaggcc 2880acggccgact tgtaagcagt ctttttgtaa ctcttagctt gcagataaaa acttttaggt 2940ttctggtact cattatttat gcatctcttg aatcacctta tctagttgta caatgacttc 3000cgaaacaacc ccaacgaccc ggtttttgct gaggtgccag ccgagcttgc tactttggac 3060atgaagaaaa gtctttcaga caagaatgtc gagaaatctc tgattgctgc ctccaagtct 3120tctttcaaca agcaggttga gcctggaatg accaccgtcc gacagctcgg aaacttgtac 3180accgcctctc tcttcggtgc tctcgcaagt ttgttctcta atgttcctgg tgacgagctc 3240gtaagtcttg atctctatcc caatcatctc ttccttatca attgaactga actcttttct 3300ttaatgctgg ctttctcttg aacaggtcgg caagcgcatt gctctctacg cctacggatc 3360tggagctgct gcttctttct atgctcttaa ggtcaagagc tcaaccgctt tcatctctga 3420gaagcttgat ctcaacaacc gattgagcaa catgaagatt gtcccctgtg atgactttgt 3480caaagctctg aaggtacgtt ggataatgac tttttttgtg gaccgtggtc tttgtcaacc 3540gctaacaacc ttcttgaatc ggtctctttt ggtttgaaat tcgctcggcg cttcgacaca 3600ggtccgagaa gagactcaca acgccgtgtc atattcgccc atcggttcgc ttgacgatct 3660ctggcctgga tcgtactact tgggagagat tgacagcatg tggcgtcgac agtacaagca 3720ggtcccttct gcttgaacgg gatattaaaa gtttcaaaag ttatgaaaga ggtcggcgaa 3780gattcaaaat aaataaatat aacaccttgc tttttggctt gttttccttc ttcactctcg 3840tttccgatgt gtttcctccg tttcttccct cttttgttcc tttttcctcc ctcttttggt 3900tacaatctct ttgggtttta caggctggca atctctgtac aatcttcgtt cgcgtgatcc 3960gacatagata ccgttgtggc atacaccttg cgtcttacat cttttgagag cttcggaggt 4020gatcttgatg aagaaaattc accattgact cccatctctt gaatgtcctg actaaattga 4080attggaagca acttatatga agagcaaatt gatggatcca gaaaggaaca agtctagaaa 4140tcagtgattt gtgcgaaaaa tcagcaaatg ccgcgctgag ccgctcgctg gggagtagac 4200attgcccatg cgcgtgatgt tgtctgaccg ttctcctcca ttcccccact ctcaaccttc 4260ctctctttga gaatcgaaga agaaggcgaa gaaaacctga cttgatcctt tacagggtgt 4320ttcttttgtt cgtatctgag ttacttttcc tcctttcctt cctgcttgag tgaatgactg 4380atctgactcc tccgcctacc tcggcgactg ggctatatct tgaggataga atatccccct 4440gacaatccca tttctcaaga ttctttcaaa caagaaaact agttccaatc aatagatcat 4500ctgatcaacc ttgtgtgaac ataatcatct gcagaagcac tgaactgaga aagtcttcct 4560cagaggaaag agaatactag ataagatcat tcggttggga aggtaaagga atgaagtctg 4620gttctgggtt tagctctggt tccgtagggg gttcgactat agtttcttct gttcgactag 4680aaacaggaga aaccgtacat gtaaatggta tgatattctt gtctctgtat catgtcccgc 4740tcatctcttt gtttgcaagt cactctggag aattc 4775 2 6370 DNA Phaffia rhodozyma5′UTR (1043)..(1044) EXPERIMENTAL 2 ggaagacatg atggtgtggg tgtgagtatgagcgtgagcg tgggtatggg cctgggtgtg 60 ggtatgagcg gtggtggtga tggatggatgggtgggtggc gtggaggggt ccgtgcggca 120 agatgttttc tctgggtagg agcgttctgcattggggcag gagaaaaaat agtgtggtta 180 cgggagatcg tggttacatc aagccatcgtcactgtaagg ctctgtaagg ctcggttgtt 240 aagaaggtaa ccaagtgtaa tcacttggttcgcggggtga cacttaggct ctggcgatta 300 atatatctga agcagaccaa actattaacaatatactttt ggataagagg tttcaacaag 360 aatctcagct tgaggaaaac tcttatccaagaaggcgcga gggcgtcccc gttttatatc 420 aggacccctc gcgcatttgg tctgccactaaagatataca tatgacgagc ctagagaggc 480 tcgagatcac gaaaactaaa aagatgaagcatgaaccatg caaactagag catgatggaa 540 aatgggcgaa gaggcataag ggatggagggaacgaatagc ctgtaggggt aacccacgta 600 agagaacacg tgatacttaa cccgtatccctgacagtcac ggtgtttctt gagagtcagt 660 aatgtccagc tgtgacctca cgtgactaaacccgacacgt gtgcttcgac cgaggtggga 720 cgatcttttt tttgggggga gaaaccgagtgggacgatag agaggactac ggagaactgt 780 agtgaattgt agtgcgctca ctacggagagttctagttga gcaagcgatg tgattttcaa 840 tacaatcccg gactacaagc tctctaatagagctctataa tagaaggaca aaagtcgtcc 900 cactcctatc tcccgcgcgt tttaatagagaccgattgtt tttttcccta atgttttatt 960 ttctttcccc gatcggctca tttttcttctctccgcgtat tcttcacaca acgctccctc 1020 cgatcttttt tcttcttgtt cctgttcctcttcgtctcct tccattgtct tctttccttc 1080 cttccttcct tcttgcctct agccagcttcaacagcgacg tctctctctc tctgtgtggt 1140 gatctccgac tgtagtgtct ctctcggtcactttcacgaa tcaacttcgt ttcttttctg 1200 atcgatcggt cgtctttccc tcaatccgtgcatacactca cacttacact cacacccaca 1260 cactcaaaca cgctaaataa tcagatccgtctccccttct tgatctcctt cggcttaggc 1320 aatggcttcc ttgttcggcc tccggcggtcctcaaacgag cagccgcgct ctcctctgct 1380 catccaatcg aagtcatcct ttctacctttgtcgtggtca ccttgacgta ctttcagttg 1440 atgtacacca tcaagcacag taatttgtacgtccgatcat ctatttgtcg tgttctcctt 1500 agtctctttc tcttcctcct ttgtctttcgcgtcagcgtg gctggatttc cgtctccatg 1560 tcatttccct tatttcctct tcctgtcatttgttcctcta cttttctttc tctacctcct 1620 ttccctgtcg tttgctttcc ttcgccagttgaccaccgat cctcaggatt catggctaac 1680 atgcccaaca caaacttgca tatcatctctcttcgtccac agtctttctc agacgattag 1740 cacacaatct accaccagct gggtcgtcgatgcgttcttc tctttgggat ccagatacct 1800 tgacctcgcg aaggttagtc agttgaccctctcatgcttc ttttctctca gtcttgtgtg 1860 tgcgcatata cccactcata gacatcttcgtacgctgcac tttccctccc ttagcaagca 1920 gactcggccg atatctttat ggtcctcctcggttacgtcc ttatgcacgg cacattcgtc 1980 cgactgttcc tcaactttcg tcggatgggcgcaaactttt ggctgccagg catggttctt 2040 gtctcgtcct cctttgcctt cctcaccgccctcctcgccg cctcgatcct caacgttccg 2100 atcgacccga tctgtctctc ggaagcacttcccttcctcg tgctcaccgt cggatttgac 2160 aaggacttta ccctcgcaaa atctgtgttcagctccccag aaatcgcacc cgtcatgctt 2220 agacgaaagc cggtgatcca accaggagatgacgacgatc tcgaacagga cgagcacagc 2280 agagtggccg ccaacaaggt tgacattcagtgggcccctc cggtcgccgc ctcccgtatc 2340 gtcattggct cggtcgagaa gatcgggtcctcgatcgtca gagactttgc cctcgaggtc 2400 gccgtcctcc ttctcggagc cgccagcgggctcggcggac tcaaggagtt ttgtaagctc 2460 gccgcgttaa ttttggtggc cgactgctgcttcaccttta ccttctatgt cgccatcctc 2520 accgtcatgg tcgaggtaag ccttttcttcaagtttcttg ctgtcatttt cctttcgaca 2580 cgtatgctca tctttcgttt ccgtctctctcacctttcca ggttcaccga atcaagatca 2640 tccggggctt ccgaccggcc cacaataaccgaacaccgaa tactgtgccc tctaccccta 2700 ctatcgacgg tcaatctacc aacagatccggcatctcgtc agggcctccg gcccgaccga 2760 ccgtgcccgt gtggaagaaa gtctggaggaagctcatggg cccagagatc gattgggcgt 2820 ccgaagctga ggctcgaaac ccggttccaaagttgaagtt gctcttagta agtaaacttc 2880 ctttgttctt ctcatcattc tttatctccgaatcctgacg tcggaccctt ctcgattcaa 2940 agatcttggc ctttcttatc cttcatatcctcaacctttg cacgcctctg accgagacca 3000 cagctatcaa gcgatcgtct agcatacaccagcccattta tgccgaccct gctcatccga 3060 tcgcacagac aaacacgacg ctccatcgggcgcacagcct agtcatcttt gatcagttcc 3120 ttagtgactg gacgaccatc gtcggagatccaatcatgag caagtggatc atcatcaccc 3180 tgggcgtgtc catcctgctg aacgggttcctcctaaaagg gatcgcttct ggctctgctc 3240 tcggacccgg tcgtgccgga ggaggaggagctgccgccgc cgccgccgtc ttgctcggag 3300 cgtgggaaat cgtcgattgg aacaatgagacagagacctc aacgaacact ccggctggtc 3360 cacccggcca caagaaccag aatgtcaacctccgactcag tctcgagcgg gatactggtc 3420 tcctccgtta ccagcgtgag caggcctaccaggcccagtc tcagatcctc gctcctattt 3480 caccggtctc tgtcgcgccc gtcgtctccaacggtaacgg taacgcatcg aaatcgattg 3540 agaaaccaat gcctcgtttg gtggtccctaacggaccaag atccttgcct gaatcaccac 3600 cttcgacgac agaatcaacc ccggtcaacaaggttatcat cggtggaccg tccgacaggc 3660 ctgccctaga cggactcgcc aatggaaacggtgccgtccc ccttgacaaa caaactgtgc 3720 ttggcatgag gtcgatcgaa gaatgcgaagaaattatgaa gagtggtctc gggccttact 3780 cactcaacga cgaagaattg attttgttgactcaaaaggg aaagattccg ccgtactcgc 3840 tggaaaaagc attgcagaac tgtgagcgggcggtcaagat tcgaagggcg gttatctgta 3900 ggtctttttc tcctttgaat ttcaagccttggaggagagg aaagtgcttc ggggtacaat 3960 acaggttgtg caaacaaacc aagagaaactaaagaaaact ttcttctcct ctctctcccc 4020 tcgacgtcag cccgagcatc cgttactaagacgctggaaa cctcggactt gcccatgaag 4080 gattacgact actcgaaagt gatgggcgcatgctgtgaga acgttgtcgg atatatgcct 4140 ctccctgtcg gaatcgctgg tccacttaacattgatggcg aggtcgtccc catcccgatg 4200 gccaccaccg agggaactct cgtggcctcgacgtcgagag gttgcaaagc gctcaacgcg 4260 ggtggcggag tgaccaccgt catcacccaggatgcgatga cgagaggacc ggtggtggat 4320 ttcccttcgg tctctcaggc cgcacaggccaaacgatggt tggattcggt cgaaggaatg 4380 gaggttatgg ccgcttcgtt caactcgacttctagattcg ccaggttgca gagcatcaag 4440 tgtggaatgg ccggccgatc gctatacatccgtttggcga ccagtaccgg agatgcgatg 4500 ggaatgaaca tggctggtga gtgcgacgagttttctttgt tcttcttgtg cggaccatgt 4560 tttctcatcc agccaattca ttcttcattccttctcggtg tttggcaacc ttttaggtaa 4620 aggaacggag aaagctttgg aaaccctgtccgagtacttc ccatccatgc agatccttgc 4680 tctttctggt aactactgta tcgacaagaagccttctgcc atcaactgga ttgagggccg 4740 tggaaagtcc gtggtggccg agtcggtgatccctggagcg atcgtcaagt ctgtcctcaa 4800 gacaacggtt gcggatctcg tcaacttgaacattaagaaa aacttgatcg gaagtgccat 4860 ggcaggcagc attggaggat tcaacgcccacgcgtcgaat attttgactg tgcgtacttc 4920 tctttccata ttcgtcctcg tttaatttcttttctgtcca gtcttatgac gtctgattgg 4980 ttcttctttt cacccacaca catacagtcaatcttcttgg ctacaggtca ggatcctgca 5040 cagaatgtgg agtcctcaat gtgcatgacattgatggagg cgtacgtttt ttgttttgtt 5100 ttccttcttt ttccatatgt ttctacttctactttcttcc cgagtccgcc aagctgatac 5160 ctttatacgg tccttctctt tctcatgacgagtagtgtga acgacggaaa agatctactc 5220 atcacctgct cgatgccggc gatcgagtgcggaacggtcg gtggaggaac tttcctccct 5280 ccgcaaaacg cctgtttgca gatgctcggtgtcgcaggtg cccatccaga ttcgcccggt 5340 cacaatgctc gtcgactagc aagaatcatcgctgccagtg tgatggctgg agagttgagt 5400 ttgatgagtg ctttggccgc tggtcatttaatcaaggccc acatgagtaa gtctgccacc 5460 ttttgataat caaaagggtc gtggtactggtgtcactgac tggtgactct tcctgtcatg 5520 cagagcacaa tcgatcgaca ccttcgactcctctaccggt ctcaccgttg gcgacccgac 5580 cgaacacgcc gtcccaccgg tcgattggattgctcacacc gatgacgtct tccgcatcgg 5640 tcgcctcgat gttctctggg ttcggtagtccgtcgacgag ctcgctcaag acggtaggta 5700 gcatggcttg cgtcagggaa cgaggggacgagacgagtgt gaacgtggat gcctgaactg 5760 gggactccct tttcttggta tcccttccgtttttctttcg gcctttgaat cctgtattct 5820 tgtccgtttt ttcatcttct cttcctggttctccttctct cgttcatctg caaaaacaaa 5880 attcaatcgc atcggtctct ggcattccatttgggtttca aaatcaaatc aatctctatc 5940 tactatctca aatatctttt tttcatcttttgattcattt ctgttgaaaa ctgtcttgcc 6000 cttctcctac ttcttatctc tgccttcttgccaaagttca attcgttgtc catctgtgca 6060 ctctgatcta tcagtctgta tcaagtacgctcttaaatct gtaattggct ctcggaggtg 6120 tctcgtcatc tcacatatgg ctggcgatatgatgtgtcgg tttcttcccc tccaacaaag 6180 gcgacgtggc tccttcatca atctttggcgcaagctctca aaattctcca aaacggctga 6240 ctaagcaagg tttccaagta ctctcaaaccgagcaaggcc atccatcctc aaatcaactt 6300 gtgaaaccct ttgtggatag accgtccaaaccgagctctt cccaatcttc gcctcccctt 6360 cttcctgcag 6370 3 4135 DNA Phaffiarhodozyma 5′UTR (911)..(912) EXPERIMENTAL 3 actgactcgg ctaccggaaaatatcttttc aggacgcctt gatcgttttg gacaacacca 60 tgatgtcacc atatcttcagcggccgttgg agctaggagt agacattgta tacgactctg 120 gaacaaagta tttgagtggacaccacgatc tcatggctgg tgtgattact actcgtactg 180 aggagattgg gaaggttcgtgcttgcttgc tttgaatgtc gtgcctaaag ccattgccat 240 aagacagagt ctgatctatgtcgtttgcct acaacagaga atggcctggt tcccaaatgc 300 tatgggaaat gcattgtctccgttcgactc gttccttctt ctccgaggac tcaaaacact 360 tcctctccga ctggacaagcagcaggcctc atctcacctg atcgcctcgt acttacacac 420 cctcggcttt cttgttcactaccccggtct gccttctgac cctgggtacg aacttcataa 480 ctctcaggcg agtggtgcaggtgccgtcat gagctttgag accggagata tcgcgttgag 540 tgaggccatc gtgggcggaacccgagtttg gggaatcagt gtcagtttcg gagccgtgaa 600 cagtttgatc agcatgccttgtctaatgag gttagttctt atgccttctt ttcgcgcctt 660 ctaaaatttc tggctgactaattgggtcgg tctttccgtt cttgcatttc agtcacgcat 720 ctattcctgc tcaccttcgagccgagcgag gtctccccga acatctgatt cgactgtgtg 780 tcggtattga ggaccctcacgatttgcttg atgatttgga ggcctctctt gtgaacgctg 840 gcgcaatccg atcagtctctacctcagatt catcccgacc gctcactcct cctgcctctg 900 attctgcctc ggacattcactccaactggg ccgtcgaccg agccagacag ttcgagcgtg 960 ttaggccttc taactcgacagccggcgtcg aaggacagct tgccgaactc aatgtagacg 1020 atgcagccag acttgcgggcgatgagagcc aaaaagaaga aattcttgtc agtgcaccgg 1080 gaaaggtcat tctgttcggcgaacatgctg taggccatgg tgttgtgagt gagaaatgaa 1140 agctttatgc tctcattgcatcttaacttt tcctcgcctt ttttgttctc ttcatcccgt 1200 cttgattgta gggatgcccccctttgcccc tttccccttc ttgcatctgt ctatatttcc 1260 ttatacattt cgctcttaagagcgtctagt tgtaccttat aacaaccttt ggttttagca 1320 tcctttgatt attcatttctctcatccttc ggtcagaggc tttcggccat ctttacgtct 1380 gattagattg taatagcaagaactatcttg ctaagccttt tctcttcctc ttcctcctat 1440 ataaatcgaa ttcactttcggacatgttta ttttggggaa atcatcaagg ggtggggggc 1500 caatcccgac actaattttctgctcacgtc aaaactcagc gttcagaatc agtcactgac 1560 cctgatacgt gtctctatgtgtgtgggtgt acgtgcgaat tgtgactcga cgttctacgc 1620 ttaaaaacag accgggatcgctgcttccgt tgatcttcga tgctacgctc ttctctcacc 1680 cactgctacg acaacaacatcatcgtcgtt atcgtctaca aacattacca tctccctaac 1740 ggacctgaac tttacgcagtcttggcctgt tgattctctt ccttggtcac ttgcgcctga 1800 ctggactgag gcgtctattccagaatctct ctgcccgaca ttgctcgccg aaatcgaaag 1860 gatcgctggt caaggtggaaacggaggaga aagggagaag gtggcaacca tggcattctt 1920 gtatttgttg gtgctattgagcaaagggaa gccaaggtag gttttttctg tctcttcttt 1980 ttgcctataa agactcttaactgacggaga aagtgttggg tttcttcctt cgggggttca 2040 atcaattaaa gtgagccgttcgagttgacg gctcgatctg cgcttccgat gggagctggt 2100 ctgggttcat ccgccgctctatcgacctct cttgccctag tctttcttct ccacttttct 2160 cacctcagtc caacgacgactggcagagaa tcaacaatcc cgacggccga cacagaagta 2220 attgacaaat gggcgttcttagctgaaaaa gtcatccatg gaaatccgag tgggattgat 2280 aacgcggtca gtacgagaggaggcgctgtt gctttcaaaa gaaagattga gggaaaacag 2340 gaaggtggaa tggaagcgatcaagaggtac gcagacacgg tgcttcatat gccatactcc 2400 agtctgattg acccatgatgaacgtctttc tacatttcga atatagcttc acatccattc 2460 gattcctcat cacagattctcgtatcggaa gggatacaag atctctcgtt gcaggagtga 2520 atgctcgact gattcaggagccagaggtga tcgtcccttt gttggaagcg attcagcaga 2580 ttgccgatga ggctattcgatgcttgaaag attcagagat ggaacgtgct gtcatgatcg 2640 atcgacttca agttagttcttgttcctttc aagactcttt gtgacattgt gtcttatcca 2700 tttcatcttc ttttttcttccttcttctgc agaacttggt ctccgagaac cacgcacacc 2760 tagcagcact tggcgtgtcccacccatccc tcgaagagat tatccggatc ggtgctgata 2820 agcctttcga gcttcgaacaaagttgacag gcgccggtgg aggtggttgc gctgtaaccc 2880 tggtgcccga tggtaaagtctctccttttc tcttccgtcc aagcgacaca tctgaccgat 2940 gcgcatcctg tacttttggtcaaccagact tctcgactga aacccttcaa gctcttatgg 3000 agacgctcgt tcaatcatcgttcgcccctt atattgcccg agtgggtggt tcaggcgtcg 3060 gattcctttc atcaactaaggccgatccgg aagatgggga gaacagactt aaagatgggc 3120 tggtgggaac ggagattgatgagctagaca gatgggcttt gaaaacgggt cgttggtctt 3180 ttgcttgaac gaaagataggaaacggtgat tagggtacag atcctttgct gtcattttta 3240 caaaacactt tcttatgtcttcatgactca acgtatgccc tcatctctat ccatagacag 3300 cacggtacct ctcaggtttcaatacgtaag cgttcatcga caaaacatgc ggcacacgaa 3360 aacgagtgga tataagggagaagagagata ttagagcgaa aaagagaaga gtgagagagg 3420 aaaaaaataa ccgagaacaacttattccgg tttgttagaa tcgaagatcg agaaatatga 3480 agtacatagt ataaagtaaagaagagaggt ttacctcaga ggtgtgtacg aaggtgagga 3540 caggtaagag gaataattgactatcgaaaa aagagaactc aacagaagca ctgggataaa 3600 gcctagaatg taagtctcatcggtccgcga tgaaagagaa attgaaggaa gaaaaagccc 3660 ccagtaaaca atccaaccaacctcttggac gattgcgaaa cacacacacg cacgcggaca 3720 tatttcgtac acaaggacgggacattcttt ttttatatcc gggtggggag agagagggtt 3780 atagaggatg aatagcaaggttgatgtttt gtaaaaggtt gcagaaaaag gaaagtgaga 3840 gtaggaacat gcattaaaaacctgcccaaa gcgatttata tcgttcttct gttttcactt 3900 ctttccgggc gctttcttagaccgcggtgg tgaagggtta ctcctgccaa ctagaagaag 3960 caacatgagt caaggattagatcatcacgt gtctcatttg acgggttgaa agatatattt 4020 agatactaac tgcttcccacgccgactgaa aagatgaatt gaatcatgtc gagtggcaac 4080 gaacgaaaga acaaatagtaagaatgaatt actagaaaag acagaatgac tagaa 4135 4 2767 DNA Phaffia rhodozyma5′UTR (372)..(373) EXPERIMENTAL 4 gaattcttcc cgactgggct gatcgacttgactggaagat ctaaggcgga gggatgaagg 60 aagtaattgg agggaatgag gaaaaaaaaaggcgagggaa cgcggtcttc tttcctggca 120 aggcaatgtc gtgtatctct cttgattctttcgttgtatc gacggaccac actcttttcg 180 aatgaatatc actatcgcat ccaatgatcgctatacatgg catttacata tgccagacat 240 cgctgagaaa gagagaacat tcctttggaaaaagcctact gtgcctgaag tcaggctgat 300 gttgattaaa cgtctttccc catcctaagcagacaaacaa cttcttttcg ttcaacacac 360 cacctctctc cgaaaaagct cttcaatccagtccattaag atggttcata tcgctactgc 420 ctcggctccc gttaacattg cgtgtatcaaggtccgtctg cattgtgaat gctgctcgtt 480 tgccttgtgt gcgtttggtg gatctgaaagaacccttgct tgaaccattc catctctgct 540 ctttttcttc ctgtcctttc ctttttctcacgacaaaaaa accacctgga ccctttgtgt 600 tcctttccat tggtgttcat acacctaacacagtactggg gtaaacggga taccaagttg 660 attctcccta caaactcctc cttgtctgtcactctcgacc aggatcacct ccgatcgacg 720 acgtcttctg cttgtgacgc ctcgttcgagaaggatcgac tttggcttaa cgggatcgag 780 gaggaggtca aggctggtgg tcggttggatgtctgcatca aggagatgaa gaagcttcga 840 gcgcaagagg aagagaagga tgccggtctggagaaagtga gtttttctcc tgtgtgcgtg 900 tgtactctgt ataggtaccg ttgacaggacagtctttctg aagagtttgg atcttactct 960 tttttggggg ggtggtggtg tttgaaataatgaccaaaat aaagctctca tctttcaacg 1020 tgcaccttgc gtcttacaac aacttcccgactgccgctgg acttgcttcc tccgcttccg 1080 gtctagctgc gttggtcgcc tcgctcgcctcgctctacaa cctcccaacg aacgcatccg 1140 aactctcgct catcgcccga caaggttctggttctgcctg ccgatcgctc ttcggcgggt 1200 tcgttgcttg ggaacagggc aagctttcctctggaaccga ctcgttcgct gttcaggtcg 1260 agcccaggga acactggccc tcactccacgcgctgatctg tgtagtttcc gacgagaaaa 1320 agacgacggc ctcgacggca ggcatgcaaaccacggtgaa cacctcgcct ttgctccaac 1380 accgaatcga acacgtcgtt ccagcccggatggaggccat cacccaggcg atccgggcca 1440 aggatttcga ctcgttcgca aagatcaccatgaaggactc caaccagttc cacgccgtct 1500 gcctcgattc ggaacccccg atcttttacttgaacgatgt ctcccgatcg atcatccatc 1560 tcgtcaccga gctcaacaga gtgtccgtccaggccggcgg tcccgtcctt gccgcctaca 1620 cgttcgacgc cgggccgaac gcggtgatctacgccgagga atcgtccatg ccggagatca 1680 tcaggttaat cgagcggtac ttcccgttgggaacggcttt cgagaacccg ttcggggtta 1740 acaccgaagg cggtgatgcc ctgagggaaggctttaacca gaacgtcgcc ccggtgttca 1800 ggaagggaag cgtcgcccgg ttgattcacacccggatcgg tgatggaccc aggacgtatg 1860 gcgaggagga gagcctgatc ggcgaagacggtctgccaaa ggtcgtcaag gcttagacta 1920 taggttgttt cttctaaatt tgagccttcctcccgcctcc cttccacaag cataaaacaa 1980 aggataaaca aatgaattat caaaataactataggttgtt tcttctaaat ttgagccttc 2040 ctcccgcctc ccttccacaa gcataaaacaaaggataaac aaatgaatta tcaaaataaa 2100 ataaaaagtc tgccttcttt gttttggaatacatcttctt tgggacatga cccttctcct 2160 tcttttccgt atacatcttt ttgggtatttcatggtgatc aaacaacatt gtgatcgaaa 2220 gcagagacgg ccatggtgct ggctttgagcgtctggcgtt ttgtgtgtcc tgcacttgag 2280 caaccccaag ctgaccgcta ggaaaactcattgatgtgat ttatatcgta cgatgaaaga 2340 gaataaaatg atagaagaac aaagaagaacaaagtagaag aacgtctgag aagaaagaca 2400 ggaaaatgac acgtacatag tgttcgatgatgaatgatat aatattaaat ataaaatgag 2460 gtaaacgtat agcatcacgg gatgaacggatgaacatgta gtggacaagg ttgggaaata 2520 ggaatgtaga atccaagaat cgttgactgatggacggacg tatgtaaaca ggtacacccc 2580 aaagaaaaga aagaaagaaa gaaagaaaacacaaagccaa ggaagtaaag cagatggtct 2640 tctaagaata cggcttcaaa aagacagtgaacactcgtcg tcgaggaatg acaagaaaag 2700 tgagagacta cgaaaggaag aaaccaagacgaaaagaaga acggagatcg aacggacaga 2760 aataaag 2767 5 4092 DNA Phaffiarhodozyma 5′UTR (787)..(788) EXPERIMENTAL 5 cgcccggtat cttgccacagatgccgccgg agtgtctggc ggagtgctag gaacaacgtc 60 atctccatct gacgagcaagcgtaccacaa gctagctctt cgtctgtcag aaggacatcc 120 acgcaccttc ctggccttcggggatggcac cttctcgtcg acttcccatg gccgtgcccc 180 tggccttgtg aagatactgtttgccaagct gagcgcctcc ccgctgctcc aggtccgcaa 240 ggtccgagag tattggacgtcgaagatatg ttcaaagtgt caggcgagtt ctcgggagaa 300 aaaaaaagcg tgggctctgaaacagtgtgg aaatgtctac aaagtgagct ggatttattg 360 tgtgtgtatg tgtgtgtgtgtgtatgttct gtgttggttg ctcactgtac tctatgctct 420 ctcttagatt tggggaacagtgctgtgaac gcgtcgcgaa acatgctgca cctagccctt 480 caccagaagg agaaccagagggcgggaatg ctggtgtctg acgctgctac tgctgctacg 540 ctagccgctg aggctgaggctggcagaaac taaatccatg acccatcaga tcttggtgat 600 tcgtggtctg aggacacccaagtccaaaag ggctatatat cgaccatcat ccgttgcggt 660 cactcagtag taactaaagctatacatagg aatgttctga acttgataac cctaacacta 720 cgaaaatatc tcggaaaatagattaatttc cttctcatct caaacaaaag acacaacacc 780 atcaatcacg ctcctttcacacactctcct ttttgctctc tcgttcgaca gaaaataaca 840 tcaatagcca aatgtccactacgcctgaag agaagaaagc agctcgagca aagttcgagg 900 ctgtcttccc ggtcattgccgatgagattc tcgattatat gaagggtgaa ggcatgcctg 960 ccgaggcttt ggaatggatgaacaaggttc gtcaagggtt tcttctttat tcttctggtc 1020 tttgtttcgg tcgaactggctttcgaactt ggccttgacc ggttggatct cggttgttgc 1080 gccaaaacga tgtcgaagcaaaacttactc ttacctgttc ggtttccttc cttccgacct 1140 tctctctacc cttgcctccgatcggtctta tagaacttgt actacaacac tcccggagga 1200 aaactcaacc gaggactttccgtggtggat acttatatcc ttctctcgcc ttctggaaaa 1260 gacatctcgg aagaagagtacttgaaggcc gctatcctcg gttggtgtat cgagcttgta 1320 cgcgttttct tcattcacctttctttctcg tcttctactc tcttctctcg aactatcttc 1380 cctgcgtgtc atcctacacgaatctttata cttacatgtt ggaacatatg ccctgttctt 1440 aattcacctc ttttgtctcggatggtagct ccaagcttac ttcttggtgg ctgatgatat 1500 gatggacgcc tcaatcacccgacgaggcca accctgttgg tacaaagttg ttagtccctt 1560 cttctctttc tgtcctctttcttctgagct atgccaattc ttgattgaaa tcggtggtgc 1620 cgtccggact aatccgtttgtcgtttttat catatcttct tgcacaaaca ggagggagtg 1680 tctaacattg ccatcaacgacgcgttcatg ctcgagggag ctatctactt tttgctcaag 1740 aagcacttcc gaaagcagagctactatgtc gatctgctag agctcttcca cgatgtttgt 1800 ctctatttct tttcttcctcccctcaataa actgtatttg tgaccattct ggatcctttc 1860 ctgacgatga atcattcttcggatgagtag gttactttcc aaaccgagtt gggacagctc 1920 atcgatctgt tgaccgctcctgaggatcac gtcgatctcg acaagttctc ccttaacaag 1980 tatgcccgtc atatattcgttttgttgcat tcacgtctga ttgtcagctc cgattattga 2040 ctctgatggt gatggtattgaccacatcat gcgatgtttg actttctcgt aggcaccacc 2100 tcatcgttgt ttacaagaccgctttctatt cattctacct tcctgtcgca ctcgctatgc 2160 gaatggtggg tctctctcttcaactgttct tcctgatttt cttgaccatc tgtaacataa 2220 atccttggaa ttttgaactctatgtcatag gtcggcgtga cagatgagga ggcgtacaag 2280 cttgcgctct cgatcctcatcccgatgggt gaatactttc aagttcagga tgatgtgctc 2340 gacgcgttcg ctcctccggagatccttgga aagatcggaa ccgacatctt ggtgcgtttt 2400 cgttccttcc ttctacgttctgttttctat cttctgactc cccgtccatc atttatgctt 2460 ctgttaaaac gtattgaaacatcaaaagga caacaaatgt tcatggccta tcaaccttgc 2520 actctctctc gcctcgcccgctcagcgaga gattctcgat acttcgtacg gtcagaagaa 2580 ctcggaggca gaggccagagtcaaggctct gtacgctgag cttgatatcc agggaaagtt 2640 caacgcttat gagtatgtcatcttttttaa attttctaat tttcttttca tctcttgttc 2700 ccaagaatta ttttgtgaaagttctgggac tgaacatggt gcatcccttt gggttcactc 2760 cgcatatgtc tcccgtttgaataggcaaca gagttacgag tcgctgaaca agttgattga 2820 cagtattgac gaagagaagagtggactcaa gaaagaagtc ttccacagct tcctgggtaa 2880 ggtctataag cgaagcaagtaattctcctc tttatatgca aagggaagat tttggcggga 2940 gtgataggta ggaagagaagggagggtcat attcattagg catttctctt gcagatatag 3000 atgatcaaaa agggatatcggtcctcttct ttgttccgaa tacataataa gtcatacgaa 3060 gccgaacatg acaaaagtggttcatgagat caaacttttt gcatgatctt ctgcgatttt 3120 gtacaattct ctcgcatcctattaggatcg aaccaggaga agatgagaga aggaaaccct 3180 caccccgtca gataacaaacgagaagtctc atcacacaca cacacagatg aaagagaaaa 3240 ataaactgac gaggataacttccaatccga tttttccagc ccacgaacct tccttggtcc 3300 ccgctccggt gccttcgagtccgatcaatg gggcccaaac gcctgaagat ccaaagaacc 3360 cttgttgagg tgtatttctcgtctgagcaa tcttagatcc ttcaatttgc agtcgcgcat 3420 atataccatc aacatcatcgtcatcaccat cattgtcgtc cacaacagca ccgcaacgcc 3480 gttaatggca gggcttggacaacttgaggc ggtttctagc aggtcggacc gattggagct 3540 cgacccaggg tgcacatcaccaagacacat tctccttcaa atgagcgaac aagacataat 3600 gagggaagta gtacgctatcgaacgtcttc tcacatcccg ggttcttggc gtatcttttg 3660 gcgattcttt ttgttgaaatagaaaattga agagaaaaaa agagatccac atgatgaaga 3720 acggctctgt agattcatgctcgaaagaaa gaaagaaaga aaaagagggg aacgaacgga 3780 tctgaatctg tggccaaccaaaaagtaggc acaaagatga caacagcgcc ctcttcgaca 3840 agtctttgaa ctgcttgtggatgagacaag tcccagcaga tcaacattcc tgctttaccc 3900 catggagtat caaacacctgagaataggtc ttgcccggct gtagataatc tctggaccgt 3960 catatgcgcg aaacgatcagtacgaccgac tctactcgaa gtcgtcaaga gcacggacga 4020 gaacgaaaag aggacaaaccgctctggatg ccataaattt ctcttctcat acctctccca 4080 cccaccctca gg 4092 6467 PRT Phaffia rhodozyma 6 Met Tyr Thr Ser Thr Thr Glu Gln Arg Pro LysAsp Val Gly Ile Leu 1 5 10 15 Gly Met Glu Ile Tyr Phe Pro Arg Arg AlaIle Ala His Lys Asp Leu 20 25 30 Glu Ala Phe Asp Gly Val Pro Ser Gly LysTyr Thr Ile Gly Leu Gly 35 40 45 Asn Asn Phe Met Ala Phe Thr Asp Asp ThrGlu Asp Ile Asn Ser Phe 50 55 60 Ala Leu Asn Ala Val Ser Gly Leu Leu SerLys Tyr Asn Val Asp Pro 65 70 75 80 Lys Ser Ile Gly Arg Ile Asp Val GlyThr Glu Ser Ile Ile Asp Lys 85 90 95 Ser Lys Ser Val Lys Thr Val Leu MetAsp Leu Phe Glu Ser His Gly 100 105 110 Asn Thr Asp Ile Glu Gly Ile AspSer Lys Asn Ala Cys Tyr Gly Ser 115 120 125 Thr Ala Ala Leu Phe Asn AlaVal Asn Trp Ile Glu Ser Ser Ser Trp 130 135 140 Asp Gly Arg Asn Ala IleVal Phe Cys Gly Asp Ile Ala Ile Tyr Ala 145 150 155 160 Glu Gly Ala AlaArg Pro Ala Gly Gly Ala Gly Ala Cys Ala Ile Leu 165 170 175 Ile Gly ProAsp Ala Pro Val Val Phe Glu Pro Val His Gly Asn Phe 180 185 190 Met ThrAsn Ala Trp Asp Phe Tyr Lys Pro Asn Leu Ser Ser Glu Tyr 195 200 205 ProIle Val Asp Gly Pro Leu Ser Val Thr Ser Tyr Val Asn Ala Ile 210 215 220Asp Lys Ala Tyr Glu Ala Tyr Arg Thr Lys Tyr Ala Lys Arg Phe Gly 225 230235 240 Gly Pro Lys Thr Asn Gly Val Thr Asn Gly His Thr Glu Val Ala Gly245 250 255 Val Ser Ala Ala Ser Phe Asp Tyr Leu Leu Phe His Ser Pro TyrGly 260 265 270 Lys Gln Val Val Lys Gly His Gly Arg Leu Leu Tyr Asn AspPhe Arg 275 280 285 Asn Asn Pro Asn Asp Pro Val Phe Ala Glu Val Pro AlaGlu Leu Ala 290 295 300 Thr Leu Asp Met Lys Lys Ser Leu Ser Asp Lys AsnVal Glu Lys Ser 305 310 315 320 Leu Ile Ala Ala Ser Lys Ser Ser Phe AsnLys Gln Val Glu Pro Gly 325 330 335 Met Thr Thr Val Arg Gln Leu Gly AsnLeu Tyr Thr Ala Ser Leu Phe 340 345 350 Gly Ala Leu Ala Ser Leu Phe SerAsn Val Pro Gly Asp Glu Leu Val 355 360 365 Gly Lys Arg Ile Ala Leu TyrAla Tyr Gly Ser Gly Ala Ala Ala Ser 370 375 380 Phe Tyr Ala Leu Lys ValLys Ser Ser Thr Ala Phe Ile Ser Glu Lys 385 390 395 400 Leu Asp Leu AsnAsn Arg Leu Ser Asn Met Lys Ile Val Pro Cys Asp 405 410 415 Asp Phe ValLys Ala Leu Lys Val Arg Glu Glu Thr His Asn Ala Val 420 425 430 Ser TyrSer Pro Ile Gly Ser Leu Asp Asp Leu Trp Pro Gly Ser Tyr 435 440 445 TyrLeu Gly Glu Ile Asp Ser Met Trp Arg Arg Gln Tyr Lys Gln Val 450 455 460Pro Ser Ala 465 7 1091 PRT Phaffia rhodozyma 7 Met Tyr Thr Ile Lys HisSer Asn Phe Leu Ser Gln Thr Ile Ser Thr 1 5 10 15 Gln Ser Thr Thr SerTrp Val Val Asp Ala Phe Phe Ser Leu Gly Ser 20 25 30 Arg Tyr Leu Asp LeuAla Lys Gln Ala Asp Ser Ala Asp Ile Phe Met 35 40 45 Val Leu Leu Gly TyrVal Leu Met His Gly Thr Phe Val Arg Leu Phe 50 55 60 Leu Asn Phe Arg ArgMet Gly Ala Asn Phe Trp Leu Pro Gly Met Val 65 70 75 80 Leu Val Ser SerSer Phe Ala Phe Leu Thr Ala Leu Leu Ala Ala Ser 85 90 95 Ile Leu Asn ValPro Ile Asp Pro Ile Cys Leu Ser Glu Ala Leu Pro 100 105 110 Phe Leu ValLeu Thr Val Gly Phe Asp Lys Asp Phe Thr Leu Ala Lys 115 120 125 Ser ValPhe Ser Ser Pro Glu Ile Ala Pro Val Met Leu Arg Arg Lys 130 135 140 ProVal Ile Gln Pro Gly Asp Asp Asp Asp Leu Glu Gln Asp Glu His 145 150 155160 Ser Arg Val Ala Ala Asn Lys Val Asp Ile Gln Trp Ala Pro Pro Val 165170 175 Ala Ala Ser Arg Ile Val Ile Gly Ser Val Glu Lys Ile Gly Ser Ser180 185 190 Ile Val Arg Asp Phe Ala Leu Glu Val Ala Val Leu Leu Leu GlyAla 195 200 205 Ala Ser Gly Leu Gly Gly Leu Lys Glu Phe Cys Lys Leu AlaAla Leu 210 215 220 Ile Leu Val Ala Asp Cys Cys Phe Thr Phe Thr Phe TyrVal Ala Ile 225 230 235 240 Leu Thr Val Met Val Glu Val His Arg Ile LysIle Ile Arg Gly Phe 245 250 255 Arg Pro Ala His Asn Asn Arg Thr Pro AsnThr Val Pro Ser Thr Pro 260 265 270 Thr Ile Asp Gly Gln Ser Thr Asn ArgSer Gly Ile Ser Ser Gly Pro 275 280 285 Pro Ala Arg Pro Thr Val Pro ValTrp Lys Lys Val Trp Arg Lys Leu 290 295 300 Met Gly Pro Glu Ile Asp TrpAla Ser Glu Ala Glu Ala Arg Asn Pro 305 310 315 320 Val Pro Lys Leu LysLeu Leu Leu Ile Leu Ala Phe Leu Ile Leu His 325 330 335 Ile Leu Asn LeuCys Thr Pro Leu Thr Glu Thr Thr Ala Ile Lys Arg 340 345 350 Ser Ser SerIle His Gln Pro Ile Tyr Ala Asp Pro Ala His Pro Ile 355 360 365 Ala GlnThr Asn Thr Thr Leu His Arg Ala His Ser Leu Val Ile Phe 370 375 380 AspGln Phe Leu Ser Asp Trp Thr Thr Ile Val Gly Asp Pro Ile Met 385 390 395400 Ser Lys Trp Ile Ile Ile Thr Leu Gly Val Ser Ile Leu Leu Asn Gly 405410 415 Phe Leu Leu Lys Gly Ile Ala Ser Gly Ser Ala Leu Gly Pro Gly Arg420 425 430 Ala Gly Gly Gly Gly Ala Ala Ala Ala Ala Ala Val Leu Leu GlyAla 435 440 445 Trp Glu Ile Val Asp Trp Asn Asn Glu Thr Glu Thr Ser ThrAsn Thr 450 455 460 Pro Ala Gly Pro Pro Gly His Lys Asn Gln Asn Val AsnLeu Arg Leu 465 470 475 480 Ser Leu Glu Arg Asp Thr Gly Leu Leu Arg TyrGln Arg Glu Gln Ala 485 490 495 Tyr Gln Ala Gln Ser Gln Ile Leu Ala ProIle Ser Pro Val Ser Val 500 505 510 Ala Pro Val Val Ser Asn Gly Asn GlyAsn Ala Ser Lys Ser Ile Glu 515 520 525 Lys Pro Met Pro Arg Leu Val ValPro Asn Gly Pro Arg Ser Leu Pro 530 535 540 Glu Ser Pro Pro Ser Thr ThrGlu Ser Thr Pro Val Asn Lys Val Ile 545 550 555 560 Ile Gly Gly Pro SerAsp Arg Pro Ala Leu Asp Gly Leu Ala Asn Gly 565 570 575 Asn Gly Ala ValPro Leu Asp Lys Gln Thr Val Leu Gly Met Arg Ser 580 585 590 Ile Glu GluCys Glu Glu Ile Met Lys Ser Gly Leu Gly Pro Tyr Ser 595 600 605 Leu AsnAsp Glu Glu Leu Ile Leu Leu Thr Gln Lys Gly Lys Ile Pro 610 615 620 ProTyr Ser Leu Glu Lys Ala Leu Gln Asn Cys Glu Arg Ala Val Lys 625 630 635640 Ile Arg Arg Ala Val Ile Ser Arg Ala Ser Val Thr Lys Thr Leu Glu 645650 655 Thr Ser Asp Leu Pro Met Lys Asp Tyr Asp Tyr Ser Lys Val Met Gly660 665 670 Ala Cys Cys Glu Asn Val Val Gly Tyr Met Pro Leu Pro Val GlyIle 675 680 685 Ala Gly Pro Leu Asn Ile Asp Gly Glu Val Val Pro Ile ProMet Ala 690 695 700 Thr Thr Glu Gly Thr Leu Val Ala Ser Thr Ser Arg GlyCys Lys Ala 705 710 715 720 Leu Asn Ala Gly Gly Gly Val Thr Thr Val IleThr Gln Asp Ala Met 725 730 735 Thr Arg Gly Pro Val Val Asp Phe Pro SerVal Ser Gln Ala Ala Gln 740 745 750 Ala Lys Arg Trp Leu Asp Ser Val GluGly Met Glu Val Met Ala Ala 755 760 765 Ser Phe Asn Ser Thr Ser Arg PheAla Arg Leu Gln Ser Ile Lys Cys 770 775 780 Gly Met Ala Gly Arg Ser LeuTyr Ile Arg Leu Ala Thr Ser Thr Gly 785 790 795 800 Asp Ala Met Gly MetAsn Met Ala Gly Lys Gly Thr Glu Lys Ala Leu 805 810 815 Glu Thr Leu SerGlu Tyr Phe Pro Ser Met Gln Ile Leu Ala Leu Ser 820 825 830 Gly Asn TyrCys Ile Asp Lys Lys Pro Ser Ala Ile Asn Trp Ile Glu 835 840 845 Gly ArgGly Lys Ser Val Val Ala Glu Ser Val Ile Pro Gly Ala Ile 850 855 860 ValLys Ser Val Leu Lys Thr Thr Val Ala Asp Leu Val Asn Leu Asn 865 870 875880 Ile Lys Lys Asn Leu Ile Gly Ser Ala Met Ala Gly Ser Ile Gly Gly 885890 895 Phe Asn Ala His Ala Ser Asp Ile Leu Thr Ser Ile Phe Leu Ala Thr900 905 910 Gly Gln Asp Pro Ala Gln Asn Val Glu Ser Ser Met Cys Met ThrLeu 915 920 925 Met Glu Ala Val Asn Asp Gly Lys Asp Leu Leu Ile Thr CysSer Met 930 935 940 Pro Ala Ile Glu Cys Gly Thr Val Gly Gly Gly Thr PheLeu Pro Pro 945 950 955 960 Gln Asn Ala Cys Leu Gln Met Leu Gly Val AlaGly Ala His Pro Asp 965 970 975 Ser Pro Gly His Asn Ala Arg Arg Leu AlaArg Ile Ile Ala Ala Ser 980 985 990 Val Met Ala Gly Glu Leu Ser Leu MetSer Ala Leu Ala Ala Gly His 995 1000 1005 Leu Ile Lys Ala His Met LysHis Asn Arg Ser Thr Pro Ser Thr Pro 1010 1015 1020 Leu Pro Val Ser ProLeu Ala Thr Arg Pro Asn Thr Pro Ser His Arg 1025 1030 1035 1040 Ser IleGly Leu Leu Thr Pro Met Thr Ser Ser Ala Ser Val Ala Ser 1045 1050 1055Met Phe Ser Gly Phe Gly Ser Pro Ser Thr Ser Ser Leu Lys Thr Val 10601065 1070 Gly Ser Met Ala Cys Val Arg Glu Arg Gly Asp Glu Thr Ser ValAsn 1075 1080 1085 Val Asp Ala 1090 8 432 PRT Phaffia rhodozyma 8 LysGlu Glu Ile Leu Val Ser Ala Pro Gly Lys Val Ile Leu Phe Gly 1 5 10 15Glu His Ala Val Gly His Gly Val Thr Gly Ile Ala Ala Ser Val Asp 20 25 30Leu Arg Cys Tyr Ala Leu Leu Ser Pro Thr Ala Thr Thr Thr Thr Ser 35 40 45Ser Ser Leu Ser Ser Thr Asn Ile Thr Ile Ser Leu Thr Asp Leu Asn 50 55 60Phe Thr Gln Ser Trp Pro Val Asp Ser Leu Pro Trp Ser Leu Ala Pro 65 70 7580 Asp Trp Thr Glu Ala Ser Ile Pro Glu Ser Leu Cys Pro Thr Leu Leu 85 9095 Ala Glu Ile Glu Arg Ile Ala Gly Gln Gly Gly Asn Gly Gly Glu Arg 100105 110 Glu Lys Val Ala Thr Met Ala Phe Leu Tyr Leu Leu Val Leu Leu Ser115 120 125 Lys Gly Lys Pro Ser Glu Pro Phe Glu Leu Thr Ala Arg Ser AlaLeu 130 135 140 Pro Met Gly Ala Gly Leu Gly Ser Ser Ala Ala Leu Ser ThrSer Leu 145 150 155 160 Ala Leu Val Phe Leu Leu His Phe Ser His Leu SerPro Thr Thr Thr 165 170 175 Gly Arg Glu Ser Thr Ile Pro Thr Ala Asp ThrGlu Val Ile Asp Lys 180 185 190 Trp Ala Phe Leu Ala Glu Lys Val Ile HisGly Asn Pro Ser Gly Ile 195 200 205 Asp Asn Ala Val Ser Thr Arg Gly GlyAla Val Ala Phe Lys Arg Lys 210 215 220 Ile Glu Gly Lys Gln Glu Gly GlyMet Glu Ala Ile Lys Ser Phe Thr 225 230 235 240 Ser Ile Arg Phe Leu IleThr Asp Ser Arg Ile Gly Arg Asp Thr Arg 245 250 255 Ser Leu Val Ala GlyVal Asn Ala Arg Leu Ile Gln Glu Pro Glu Val 260 265 270 Ile Val Pro LeuLeu Glu Ala Ile Gln Gln Ile Ala Asp Glu Ala Ile 275 280 285 Arg Cys LeuLys Asp Ser Glu Met Glu Arg Ala Val Met Ile Asp Arg 290 295 300 Leu GlnAsn Leu Val Ser Glu Asn His Ala His Leu Ala Ala Leu Gly 305 310 315 320Val Ser His Pro Ser Leu Glu Glu Ile Ile Arg Ile Gly Ala Asp Lys 325 330335 Pro Phe Glu Leu Arg Thr Lys Leu Thr Gly Ala Gly Gly Gly Gly Cys 340345 350 Ala Val Thr Leu Val Pro Asp Asp Phe Ser Thr Glu Thr Leu Gln Ala355 360 365 Leu Met Glu Thr Leu Val Gln Ser Ser Phe Ala Pro Tyr Ile AlaArg 370 375 380 Val Gly Gly Ser Gly Val Gly Phe Leu Ser Ser Thr Lys AlaAsp Pro 385 390 395 400 Glu Asp Gly Glu Asn Arg Leu Lys Asp Gly Leu ValGly Thr Glu Ile 405 410 415 Asp Glu Leu Asp Arg Trp Ala Leu Lys Thr GlyArg Trp Ser Phe Ala 420 425 430 9 401 PRT Phaffia rhodozyma 9 Met ValHis Ile Ala Thr Ala Ser Ala Pro Val Asn Ile Ala Cys Ile 1 5 10 15 LysTyr Trp Gly Lys Arg Asp Thr Lys Leu Ile Leu Pro Thr Asn Ser 20 25 30 SerLeu Ser Val Thr Leu Asp Gln Asp His Leu Arg Ser Thr Thr Ser 35 40 45 SerAla Cys Asp Ala Ser Phe Glu Lys Asp Arg Leu Trp Leu Asn Gly 50 55 60 IleGlu Glu Glu Val Lys Ala Gly Gly Arg Leu Asp Val Cys Ile Lys 65 70 75 80Glu Met Lys Lys Leu Arg Ala Gln Glu Glu Glu Lys Asp Ala Gly Leu 85 90 95Glu Lys Leu Ser Ser Phe Asn Val His Leu Ala Ser Tyr Asn Asn Phe 100 105110 Pro Thr Ala Ala Gly Leu Ala Ser Ser Ala Ser Gly Leu Ala Ala Leu 115120 125 Val Ala Ser Leu Ala Ser Leu Tyr Asn Leu Pro Thr Asn Ala Ser Glu130 135 140 Leu Ser Leu Ile Ala Arg Gln Gly Ser Gly Ser Ala Cys Arg SerLeu 145 150 155 160 Phe Gly Gly Phe Val Ala Trp Glu Gln Gly Lys Leu SerSer Gly Thr 165 170 175 Asp Ser Phe Ala Val Gln Val Glu Pro Arg Glu HisTrp Pro Ser Leu 180 185 190 His Ala Leu Ile Cys Val Val Ser Asp Glu LysLys Thr Thr Ala Ser 195 200 205 Thr Ala Gly Met Gln Thr Thr Val Asn ThrSer Pro Leu Leu Gln His 210 215 220 Arg Ile Glu His Val Val Pro Ala ArgMet Glu Ala Ile Thr Gln Ala 225 230 235 240 Ile Arg Ala Lys Asp Phe AspSer Phe Ala Lys Ile Thr Met Lys Asp 245 250 255 Ser Asn Gln Phe His AlaVal Cys Leu Asp Ser Glu Pro Pro Ile Phe 260 265 270 Tyr Leu Asn Asp ValSer Arg Ser Ile Ile His Leu Val Thr Glu Leu 275 280 285 Asn Arg Val SerVal Gln Ala Gly Gly Pro Val Leu Ala Ala Tyr Thr 290 295 300 Phe Asp AlaGly Pro Asn Ala Val Ile Tyr Ala Glu Glu Ser Ser Met 305 310 315 320 ProGlu Ile Ile Arg Leu Ile Glu Arg Tyr Phe Pro Leu Gly Thr Ala 325 330 335Phe Glu Asn Pro Phe Gly Val Asn Thr Glu Gly Gly Asp Ala Leu Arg 340 345350 Glu Gly Phe Asn Gln Asn Val Ala Pro Val Phe Arg Lys Gly Ser Val 355360 365 Ala Arg Leu Ile His Thr Arg Ile Gly Asp Gly Pro Arg Thr Tyr Gly370 375 380 Glu Glu Glu Ser Leu Ile Gly Glu Asp Gly Leu Pro Lys Val ValLys 385 390 395 400 Ala 10 355 PRT Phaffia rhodozyma 10 Met Ser Thr ThrPro Glu Glu Lys Lys Ala Ala Arg Ala Lys Phe Glu 1 5 10 15 Ala Val PhePro Val Ile Ala Asp Glu Ile Leu Asp Tyr Met Lys Gly 20 25 30 Glu Gly MetPro Ala Glu Ala Leu Glu Trp Met Asn Lys Asn Leu Tyr 35 40 45 Tyr Asn ThrPro Gly Gly Lys Leu Asn Arg Gly Leu Ser Val Val Asp 50 55 60 Thr Tyr IleLeu Leu Ser Pro Ser Gly Lys Asp Ile Ser Glu Glu Glu 65 70 75 80 Tyr LeuLys Ala Ala Ile Leu Gly Trp Cys Ile Glu Leu Leu Gln Ala 85 90 95 Tyr PheLeu Val Ala Asp Asp Met Met Asp Ala Ser Ile Thr Arg Arg 100 105 110 GlyGln Pro Cys Trp Tyr Lys Val Glu Gly Val Ser Asn Ile Ala Ile 115 120 125Asn Asn Ala Phe Met Leu Glu Gly Ala Ile Tyr Phe Leu Leu Lys Lys 130 135140 His Phe Arg Lys Gln Ser Tyr Tyr Val Asp Leu Leu Glu Leu Phe His 145150 155 160 Asp Val Thr Phe Gln Thr Glu Leu Gly Gln Leu Ile Asp Leu LeuThr 165 170 175 Ala Pro Glu Asp His Val Asp Leu Asp Lys Phe Ser Leu AsnLys His 180 185 190 His Leu Ile Val Val Tyr Lys Thr Ala Phe Tyr Ser PheTyr Leu Pro 195 200 205 Val Ala Leu Ala Met Arg Met Val Gly Val Thr AspGlu Glu Ala Tyr 210 215 220 Lys Leu Ala Leu Ser Ile Leu Ile Pro Met GlyGlu Tyr Phe Gln Val 225 230 235 240 Gln Asp Asp Val Leu Asp Ala Phe ArgPro Pro Glu Ile Leu Gly Lys 245 250 255 Ile Gly Thr Asp Ile Leu Asp AsnLys Cys Ser Trp Pro Ile Asn Leu 260 265 270 Ala Leu Ser Pro Ala Ser ProAla Gln Arg Glu Ile Leu Asp Thr Ser 275 280 285 Tyr Gly Gln Lys Asn SerGlu Ala Glu Ala Arg Val Lys Ala Leu Tyr 290 295 300 Ala Glu Leu Asp IleGln Gly Lys Phe Asn Ala Tyr Glu Gln Gln Ser 305 310 315 320 Tyr Glu SerLeu Asn Lys Leu Ile Asp Ser Ile Asp Glu Glu Lys Ser 325 330 335 Gly LeuLys Lys Glu Val Phe His Ser Phe Leu Gly Lys Val Tyr Lys 340 345 350 ArgSer Lys 355 11 26 DNA Artificial Sequence Description of ArtificialSequence Degenerate sense primer for cloning of HMC 11 ggnaartayacnathggnyt nggnca 26 12 26 DNA Artificial Sequence Description ofArtificial Sequence Degenerate antisense primer for cloning of HMC gene12 tanarnswns wngtrtacat rttncc 26 13 24 DNA Artificial SequenceDescription of Artificial Sequence Primary primer for cloning of5′-adjacent region of HMC gene 13 gaagaacccc atcaaaagcc tcga 24 14 25DNA Artificial Sequence Description of Artificial Sequence Nested primerfor cloning of 5′-adjacent region of HMC gene 14 aaaagcctcg agatccttgtgagcg 25 15 18 DNA Artificial Sequence Description of ArtificialSequence Sense primer for cloning of small EcoRI portion of HMC gene 15agaagccaga agagaaaa 18 16 18 DNA Artificial Sequence Description ofArtificial Sequence Antisense primer for cloning of small EcoRI portionof HMC gene 16 tcgtcgagga aagtagat 18 17 30 DNA Artificial SequenceDescription of Artificial Sequence Sense primer for cloning of cDNA ofHMC gene 17 ggtaccatat gtatccttct actaccgaac 30 18 30 DNA ArtificialSequence Description of Artificial Sequence Antisense primer for cloningof cDNA of HMC gene 18 gcatgcggat cctcaagcag aagggacctg 30 19 32 DNAArtificial Sequence Description of Artificial Sequence Degenerate senseprimer for cloning HMG gene 19 gcntgytgyg araaygtnat hggntayatg cc 32 2032 DNA Artificial Sequence Description of Artificial Sequence Degenerateantisense primer for cloning of HMG gene 20 atccarttda tngcngcnggyttyttrtcn gt 32 21 25 DNA Artificial Sequence Description of ArtificialSequence Antisense primer for cloning of cDNA of HMG gene 21 ggccattccacacttgatgc tctgc 25 22 21 DNA Artificial Sequence Description ofArtificial Sequence Sense primer for cloning of cDNA of HMG gene 22ggccgatatc tttatggtcc t 21 23 26 DNA Artificial Sequence Description ofArtificial Sequence Sense primer for cloning of cDNA of HMG gene 23ggtaccgaag aaattatgaa gagtgg 26 24 26 DNA Artificial SequenceDescription of Artificial Sequence Antisense primer for cloning of cDNAof HMG gene 24 ctgcagtcag gcatccacgt tcacac 26 25 29 DNA ArtificialSequence Description of Artificial Sequence Degenerate sense primer forcloning of MVK gene 25 gcnccnggna argtnathyt nttyggnga 29 26 29 DNAArtificial Sequence Description of Artificial Sequence Degenerateantisense primer for cloning of MVK gene 26 ccccangtns wnacngcrttrtcnacncc 29 27 17 DNA Artificial Sequence Description of ArtificialSequence Sense primer for cloning of genomic DNA containing MVK gene 27acatgctgta gtccatg 17 28 16 DNA Artificial Sequence Description ofArtificial Sequence Antisense primer for cloning of genomic DNAcontaining MVK gene 28 actcggattc catgga 16 29 25 DNA ArtificialSequence Description of Artificial Sequence Primer for genomic walkingto clone 5′-adjacent region of MVK gene 29 ttgttgtcgt agcagtgggt gagag25 30 18 DNA Artificial Sequence Description of Artificial SequenceSense primer for cloning of 5′-adjacent region of MVK gene 30 ggaagaggaagagaaaag 18 31 18 DNA Artificial Sequence Description of ArtificialSequence Antisense primer for cloning of 5′-adjacent region of MVK gene31 ttgccgaact caatgtag 18 32 26 DNA Artificial Sequence Description ofArtificial Sequence Sense primer for introduction of a nucleotide intoMVK gene 32 ggatccatga gagcccaaaa agaaga 26 33 26 DNA ArtificialSequence Description of Artificial Sequence Antisense primer forintroduction of a nucleotide into MVK gene 33 gtcgactcaa gcaaaagaccaacgac 26 34 23 DNA Artificial Sequence Description of ArtificialSequence Degenerate sense primer for cloning of MPD gene 34 htnaartayttgggnaarmg nga 23 35 29 DNA Artificial Sequence Description ofArtificial Sequence Degenerate antisense primer for cloning of MPD gene35 gcrttnggnc cngcrtcraa ngtrtangc 29 36 20 DNA Artificial SequenceDescription of Artificial Sequence Sense primer for colony PCR to clonea genomic MPD clone 36 ccgaactctc gctcatcgcc 20 37 20 DNA ArtificialSequence Description of Artificial Sequence Antisense primer for colonyPCR to clone a genomic MPD clone 37 cagatcagcg cgtggagtga 20 38 26 DNAArtificial Sequence Description of Artificial Sequence Degenerate senseprimer for cloning of FPS gene 38 cargcntayt tyytngtngc ngayga 26 39 32DNA Artificial Sequence Description of Artificial Sequence Degenerateantisense primer for cloning of FPS gene 39 cayttrttrt cytgdatrtcngtnccdaty tt 32 40 25 DNA Artificial Sequence Description of ArtificialSequence Sense primer for cloningof FPS downstream region 40 atcctcatcccgatgggtga atact 25 41 25 DNA Artificial Sequence Description ofArtificial Sequence Antisense primer for cloning of FPS upstream region41 aggagcggtc aacagatcga tgagc 25 42 25 DNA Artificial SequenceDescription of Artificial Sequence Sense primer for cloning of cDNA andgenomic FPS gene 42 gaattcatat gtccactacg cctga 25 43 25 DNA ArtificialSequence Description of Artificial Sequence Antisense primer for cloningof cDNA and genomic FPS gene 43 gtcgacggta cctatcactc ccgcc 25

What is claimed is:
 1. An isolated DNA sequence comprising a DNAsequence that hybridizes to SEQ ID NO:2 under the following conditions:hybridization in 50% formnamide (v/v), 2% blocking agent, 5×SSC, 0.1%N-lauroylsarcosine (w/v), and 0.1% SDS at 42° C. overnight followed bytwo washes for 5 minutes each in 2×SSC and 0.1% SDS at room temperaturefollowed by two additional washes of 15 minutes each in 0.1×SSC and 0.1%SDS at 68° C., wherein the DNA sequence encodes an amino acid sequencehaving 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase)activity.
 2. An isolated DNA sequence according to claim 1 wherein theDNA sequence encodes the amino acid sequence set forth in SEQ ID NO:7.3. An isolated DNA sequence according to claim 1 comprising SEQ ID NO:2.4. An isolated DNA sequence according to claim 3 consisting essentiallyof SEQ D NO:2.
 5. An isolated DNA sequence according to claim 1consisting of SEQ ID NO:2 or a fragment thereof, which fragment encodesan amino acid sequence having HMG-CoA reductase activity.
 6. An isolatedDNA sequence according to claim 1 wherein the DNA sequence is anisocoding variant of the DNA sequence of SEQ ID NO:2.
 7. An isolated DNAsequence according to claim 1 wherein the DNA sequence that hybridizesto SEQ D NO:2 is a derivative of SEQ ID NO:2, which contains anaddition, insertion, deletion, and/or substitution of one or morenucleotide(s).
 8. An isolated DNA sequence according claim 1 wherein theDNA sequence is derived from a Phaffia rhodozyma gene and is selectedfrom the group consisting of SEQ ID NO:2, an isocoding variant of SEQ IDNO:2, and a derivative of a SEQ ID NO:2 having an addition, insertion,deletion and/or substitution of one or more nucleotide(s).
 9. A vectoror plasmid comprising a DNA sequence according to claim
 1. 10. A hostcell transformed or transfected with a DNA sequence according to claim1.
 11. A host cell transformed or transfected with a vector or plasmidaccording to claim
 9. 12. A process for producing an enzyme forconverting acetyl Co-A to isopentyl pyrophosphate, which enzyme is inthe mevalonate biosynthetic pathway or for converting isopentylpyrophosphate to farnesyl pyrophosphate, which comprises culturing ahost ing to claim 10, under conditions wherein the enzyme is produced.13. A process for the production of isoprenoids or carotenoids, whichgrowing a host cell according to clam 10 under conditions whereinisoprenoids or carotenoids are produced.
 14. A process according toclaim 13 wherein the carotenoid is astaxanthin.
 15. An isolated DNAsequence comprising a DNA sequence that does not to SEQ ID NO:2 butwhich encodes an amino acid sequence that is identical to SEQ ID NO:7.